Variants of Glycoside Hydrolases

ABSTRACT

The present invention relates to variants of a parent glycoside hydrolase, comprising a substitution at one or more positions corresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprising a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 a substitution at one or more positions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity. The present invention also relates to nucleotide sequences encoding the variant glycoside hydrolases and to nucleic acid constructs, vectors, and host cells comprising the nucleotide sequences.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No. 13/748,328filed on Jan. 23, 2013, now pending, which is a divisional of U.S.application Ser. No. 13/009,524 filed on Jan. 19, 2011, now U.S. Pat.No. 8,383,385, which is a divisional of U.S. application Ser. No.11/891,249 filed on Aug. 8, 2007, now U.S. Pat. No. 7,932,073, which isa continuation of U.S. application Ser. No. 10/926,223 filed on Aug. 25,2004, now abandoned, which claims the benefit of U.S. ProvisionalApplication No. 60/497,809 filed on Aug. 25, 2003. The content of theseapplications is incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with Government support under NREL SubcontractNo. ZCO-30017-02, Prime Contract DE-AC36-98G010337 awarded by theDepartment of Energy. The government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to variants of a glycoside hydrolasehaving one or more improved properties relative to its parent enzyme,nucleic acids encoding the variants, methods of producing the variants,and methods of using the variants.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently bonded bybeta-1,4-linkages. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of cellulosic feedstocks into ethanol has the advantagesof the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the cellulose is converted to glucose,the glucose is easily fermented by yeast into ethanol.

St∪hlberg et al., 1996, J. Mol. Biol. 264: 337-349, describe activitystudies and crystal structures of catalytically deficient mutants ofcellobiohydrolase I from Trichoderma reesei. Boer and Koivula, 2003,Eur. J. Biochem. 270: 841-848, disclose the relationship between thermalstability and pH optimum studied with wild-type and mutant Trichodermareesei cellobiohydrolase CeI7A.

WO 2004/016760 discloses variants of a Hypocrea jecorinacellobiohydrolase.

It would be an advantage in the art to provide glycoside hydrolasevariants with improved properties for converting cellulosic materials tomonosaccharides, disaccharides, and polysaccharides. Improved propertiesinclude altered temperature-dependent activity profiles,thermostability, pH activity, pH stability, substrate specificity,product specificity, and chemical stability.

It is an object of the present invention to provide variants ofglycoside hydrolases with improved properties compared to its parentenzyme.

SUMMARY OF THE INVENTION

The present invention relates to isolated variants of a parent glycosidehydrolase, comprising a substitution at one or more positionscorresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373,383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprising a substitution at one or more positionscorresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227,246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513of SEQ ID NO: 2, wherein the variants have glycoside hydrolase activity.

The present invention also relates to isolated polypeptides havingglycoside hydrolase activity, wherein the amino acid sequences of thepolypeptides differ from amino acids 1 to 513 of SEQ ID NO: 2 at one ormore positions corresponding to positions 21, 94, 157, 205, 206, 247,337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 ofSEQ ID NO: 2, and optionally further differs at one or more positionscorresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227,246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513of SEQ ID NO: 2.

The present invention also relates to isolated nucleotide sequencesencoding the variant glycoside hydrolases or polypeptides havingglycoside hydrolase activity and to nucleic acid constructs, vectors,and host cells comprising the nucleotide sequences.

The present invention also relates to methods for producing variants ofa parent glycoside hydrolase or polypeptides having glycoside hydrolaseactivity in a host cell.

The present invention also relates to methods for obtaining a variant ofa parent glycoside hydrolase, comprising:

(a) introducing a substitution at one or more positions corresponding topositions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467,and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally furtherintroducing a substitution at one or more positions corresponding topositions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255,259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO:2, wherein the variant has glycoside hydrolase activity; and

(b) recovering the variant.

The present invention further relates to methods of using the glycosidehydrolase variants in detergents and in the conversion of cellulose toglucose.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of pAJ052.

FIG. 2 shows a restriction map of pJC106.

FIG. 3 shows a restriction map of pAlLo1.

FIG. 4 shows a restriction map of pBANe10.

FIG. 5 shows a restriction map of pAlLo2.

FIG. 6 shows a restriction map of pCW026.

FIG. 7 shows a restriction map of pNP776G205R.

FIG. 8 shows a restriction map of pMJ04.

FIG. 9 shows a restriction map of pMJ06.

FIG. 10 shows a restriction map of pMJ09.

FIG. 11 shows a restriction map of pCW045.

FIG. 12 shows a restriction map of pSTM01.

FIG. 13 shows a restriction map of pSMKO3.

FIG. 14 shows a restriction map of pEJG97.

FIGS. 15A and 15B show the genomic DNA sequence and the deduced aminoacid sequence of an Aspergillus fumigatus be'ta-glucosidase (SEQ ID NOS:56 and 57, respectively). The predicted signal peptide is underlined andpredicted introns are italicized.

FIG. 16 shows the thermal stability of Aspergillus fumigatusbeta-glucosidase at 50° and 65° C.

FIG. 17 shows the thermal stability of Aspergillus fumigatusbeta-glucosidase at 70° C.

FIG. 18 shows the hydrolysis of cellobiose by Aspergillus fumigatusbeta-glucosidase at 65° C.

FIG. 19 shows the time course profiles of PCS hydrolysis by the parentTrichoderma reesei strain RutC30 and the strain expressing variant776-M57.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to isolated variants of a parent glycosidehydrolase, comprising a substitution at one or more positionscorresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373,383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprising a substitution at one or more positionscorresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227,246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513of SEQ ID NO: 2, wherein the variant has glycoside hydrolase activity.

DEFINITIONS

The term “glycoside hydrolase” is defined herein as hydrolases describedby Coutinho, P. M. and Henrissat, B., 1999, Carbohydrate-active enzymes:an integrated database approach, in “Recent Advances in CarbohydrateBioengineering”, H. J. Gilbert, G. Davies, B. Henrissat and B. Svenssoneds., The Royal Society of Chemistry, Cambridge, pp. 3-12. Examples ofglycoside hydrolases include, but are not limited to, cellobiohydrolase,endoglucanase, and exoglucanase. In a preferred embodiment, theglycoside hydrolases belong to Family 7 as defined by Coutinho, P. M.and Henrissat, B., 1999, supra.

The term “cellobiohydrolase” is defined herein as a 1,4-D-glucancellobiohydrolase (E.C. 3.2.1.91) which catalyzes the hydrolysis of1,4-beta-D-glucosidic linkages in cellulose, cellotetriose, or anybeta-1,4-linked glucose containing polymer, releasing cellobiose fromthe non-reducing ends of the chain. For purposes of the presentinvention, cellobiohydrolase activity is determined according to theprocedures described by Lever et al., 1972, Anal. Biochem. 47: 273-279and by van Tilbeurgh et al., 1982, FEBS Letters, 149: 152-156; vanTilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288. In thepresent invention, the Lever et al. method was employed to assesshydrolysis of cellulose in corn stover, while the method of vanTilbeurgh et al. was used to determine the cellobiohydrolase activity ona fluorescent disaccharide derivative.

The term “endoglucanase” is defined herein as anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. No. 3.2.1.4)which catalyses endohydrolysis of 1,4-beta-D-glycosidic linkages incellulose, cellulose derivatives (such as carboxy methyl cellulose andhydroxy ethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3glucans such as cereal beta-D-glucans or xyloglucans, and other plantmaterial containing cellulosic components. For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) hydrolysis according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268.

The term “exoglucanase” is defined herein as a 1,4-beta-D-glucanglucohydrolase (E.C. 3.2.1.74) which catalyzes the hydrolysis of1,4-linkages (o-glycosyl bonds) in 1,4-beta-D-glucans so as to removesuccessive glucose or cellobiose units. For purposes of the presentinvention, exoglucanase activity is determined according to theprocedure described by Himmel et al., 1986, J. Biol. Chem. 261:12948-12955.

Variant: The term “variant” is defined herein as a glycoside hydrolasecomprising one or more alterations, such as substitutions, insertions,deletions, and/or truncations of one or more specific amino acidresidues at one or more specific positions in the polypeptide.

Wild-Type Enzyme: The term “wild-type” glycoside hydrolase denotes aglycoside hydrolase expressed by a naturally occurring microorganism,such as a yeast or a filamentous fungus found in nature.

Parent Enzyme: The term “parent” glycoside hydrolase as used hereinmeans a glycoside hydrolase to which modifications, e.g.,substitution(s), insertion(s), deletion(s), and/or truncation(s), aremade to produce the enzyme variants of the present invention. This termalso refers to the polypeptide with which a variant is compared andaligned. The parent may be a naturally occurring (wild type)polypeptide, or it may even be a variant thereof, prepared by anysuitable means. For instance, the parent protein may be a variant of anaturally occurring polypeptide which has been modified or altered inthe amino acid sequence. A parent may also be an allelic variant whichis a polypeptide encoded by any of two or more alternative forms of agene occupying the same chromosomal locus.

Shuffling: The term “shuffling” means recombination of nucleotidesequence(s) between two or more homologous nucleotide sequencesresulting in recombined nucleotide sequences (i.e., nucleotide sequenceshaving been subjected to a shuffling cycle) having a number ofnucleotides exchanged, in comparison to the starting nucleotidesequences.

Randomized library: The term “randomized library”, “variant library”, or“library” is defined herein as a library of variant polypeptides.Diversity in the variant library can be generated via mutagenesis of thegenes encoding the variants at the DNA triplet level, such thatindividual codons are variegated, e.g., by using primers of partiallyrandomized sequences in a PCR reaction. Several techniques have beendescribed, by which one can create a diverse combinatorial library byvariegating several nucleotide positions in a gene and recombining them,for example, where these positions are too far apart to be covered by asingle (spiked or doped) oligonucleotide primer. These techniquesinclude the use of in vivo recombination of the individually diversifiedgene segments as described in WO 97/07205 on page 3, lines 8 to 29. Theyalso include the use of DNA shuffling techniques to create a library offull length genes, wherein several gene segments are combined, andwherein each segment may be diversified, e.g., by spiked mutagenesis(Stemmer, 1994, Nature 370: 389-391; U.S. Pat. No. 5,811,238; U.S. Pat.No. 5,605,793; and U.S. Pat. No. 5,830,721). One can use a gene encodinga protein “backbone” (wild type parent polypeptide) as a templatepolynucleotide, and combine this with one or more single ordouble-stranded oligonucleotides as described in WO 98/41623 and WO98/41622. The single-stranded oligonucleotides can be partiallyrandomized during synthesis. The double-stranded oligonucleotides can bePCR products incorporating diversity in a specific region. In bothcases, one can dilute the diversity with corresponding segments encodingthe sequence of the backbone protein in order to limit the averagenumber of changes that are introduced.

Recombination: The term “recombination” is defined herein as a processwherein nucleic acids associate with each other in regions of homology,leading to interstrand DNA exchange between those sequences. Forpurposes of the present invention, homologous recombination isdetermined according to the procedures summarized by Paques and Haber,1999, Microbiology and Molecular Biology Reviews 63: 349-404.“Homologous recombination” is defined herein as recombination in whichno changes in the nucleotide sequences occurs within the regions ofhomology relative to the input nucleotide sequences. For perfecthomologous recombination, the regions should contain a sufficient numberof nucleic acids, such as 15 to 1,500 base pairs, preferably 100 to1,500 base pairs, more preferably 400 to 1,500 base pairs, and mostpreferably 800 to 1,500 base pairs, which are highly homologous with thecorresponding nucleic acid sequence to enhance the probability ofhomologous recombination. The recombination may also occur bynon-homologous recombination. “Non-homologous recombination” is definedherein as recombination where any mode of DNA repair incorporatingstrand exchange results in a nucleotide sequence different from any ofthe recombining sequences.

Improved property: The term “improved property” is defined herein as acharacteristic associated with a variant which is improved compared tothe parent glycoside hydrolase. Such improved properties include, butare not limited to, altered temperature-dependent activity profile,thermostability, pH activity, pH stability, substrate specificity,product specificity, and chemical stability.

Improved thermal activity: The term “improved thermal activity” isdefined herein as an alteration of the temperature-dependent activityprofile of a glycoside hydrolase variant at a specific temperaturerelative to the temperature-dependent activity profile of the parentglycoside hydrolase. The thermal activity value provides a measure ofthe enzyme's efficiency in performing catalysis of a hydrolysis reactionover a range of temperatures. A glycoside hydrolase has a specifictemperature range wherein the protein is stable and retains itsenzymatic activity, but becomes less stable and thus less active withincreasing temperature. Furthermore, the initial rate of a reactioncatalyzed by a glycoside hydrolase can be accelerated by an increase intemperature which is measured by determining thermal activity of avariant. A more thermoactive variant will lead to an increase in therate of hydrolysis decreasing the time required and/or decreasing theenzyme concentration required for hydrolysis. Alternatively, a variantwith a reduced thermal activity will catalyze a hydrolysis reaction at atemperature lower than the temperature optimum of the parent enzymedefined by the temperature-dependent activity profile of the parent.

Improved thermostability: The term “improved thermostability” is definedherein as a variant enzyme displaying retention of enzymatic activityafter a period of incubation at elevated temperature relative to theparent enzyme. Such a variant may or may not display an altered thermalactivity profile relative to the parent. For example, a variant may havean improved ability to refold following incubation at elevatedtemperature relative to the parent.

In a preferred embodiment, the thermal activity of the variant glycosidehydrolase is at least 1.5-fold, preferably at least 2-fold, morepreferably at least 5-fold, most preferably at least 7-fold, and evenmost preferably at least 20-fold more thermally active than the wildtype variant when activity on 4-methylumbelliferyl beta-D-lactoside at64° C. or a higher temperature is compared to activity at 50° C., for 45minutes at pH 5.0.

Improved product specificity: The term “improved product specificity” isdefined herein as a variant enzyme displaying an altered product profilerelative to the parent in which the altered product profile improves theperformance of the variant in a given application relative to theparent. The term “product profile” is defined herein as the chemicalcomposition of the reaction products produced by enzymatic hydrolysis.

Improved chemical stability: The term “improved chemical stability” isdefined herein as a variant enzyme displaying retention of enzymaticactivity after a period of incubation in the presence of a chemical orchemicals, either naturally occurring or synthetic, which reduce theenzymatic activity of the parent enzyme. Improved chemical stability mayalso result in variants better able to catalyze a reaction in thepresence of such chemicals.

Conventions for Designation of Variants

In the present invention, a specific numbering of amino acid residuepositions in the glycoside hydrolase variants is employed. For example,by aligning the amino acid sequences of known glycoside hydrolases, itis possible to designate an amino acid position number to any amino acidresidue in any glycoside hydrolase enzyme.

Using the numbering system originating from the amino acid sequence ofthe glycoside hydrolase disclosed in SEQ ID NO: 2, aligned with theamino acid sequence of a number of other glycoside hydrolases, it ispossible to indicate the position of an amino acid residue in aglycoside hydrolase in regions of structural homology.

Multiple alignments of protein sequences may be made, for example, using“ClustalW” (Thompson, J. D., Higgins, D. G. and Gibson, T. J., 1994,CLUSTAL W: Improving the sensitivity of progressive multiple sequencealignment through sequence weighting, positions-specific gap penaltiesand weight matrix choice, Nucleic Acids Research 22: 4673-4680).Multiple alignments of DNA sequences may be done using the proteinalignment as a template, replacing the amino acids with thecorresponding codon from the DNA sequence.

Pairwise sequence comparison algorithms in common use are adequate todetect similarities between protein sequences that have not divergedbeyond the point of approximately 20-30% sequence identity (Doolittle,1992, Protein Sci. 1: 191-200; Brenner et al., 1998, Proc. Natl. Acad.Sci. USA 95, 6073-6078). However, truly homologous proteins with thesame fold and similar biological function have often diverged to thepoint where traditional sequence-based comparisons fail to detect theirrelationship (Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615).Greater sensitivity in sequence-based searching can be attained usingsearch programs that utilize probabilistic representations of proteinfamilies (profiles) to search databases. For example, the PSI-BLASTprogram generates profiles through an iterative database search processand is capable of detecting remote homologs (Atschul et al., 1997,Nucleic Acids Res. 25: 3389-3402). Even greater sensitivity can beachieved if the family or superfamily for the protein of interest hasone or more representatives in the protein structure databases. Programssuch as GenTHREADER (Jones 1999, J. Mol. Biol. 287: 797-815; McGuffinand Jones, 2003, Bioinformatics 19: 874-881) utilize information from avariety of sources (PSI-BLAST, secondary structure prediction,structural alignment profiles, and solvation potentials) as input to aneural network that predicts the structural fold for a query sequence.Similarly, the method of Gough et al., 2000, J. Mol. Biol. 313: 903-919,can be used to align a sequence of unknown structure with thesuperfamily models present in the SCOP database. These alignments can inturn be used to generate homology models for the protein of interest,and such models can be assessed for accuracy using a variety of toolsdeveloped for that purpose.

For proteins of known structure, several tools and resources areavailable for retrieving and generating structural alignments. Forexample the SCOP superfamilies of proteins have been structurallyaligned, and those alignments are accessible and downloadable. Thesealignments can be used to predict the structurally and functionallycorresponding amino acid residues in proteins within the same structuralsuperfamily. This information, along with information derived fromhomology modeling and profile searches, can be used to predict whichresidues to mutate when moving mutations of interest from one protein toa close or remote homolog.

In describing the various glycoside hydrolase variants of the presentinvention, the nomenclature described below is adapted for ease ofreference. In all cases, the accepted IUPAC single letter or tripleletter amino acid abbreviation is employed.

Substitutions. For an amino acid substitution, the followingnomenclature is used: Original amino acid, position, substituted aminoacid. Accordingly, the substitution of threonine with alanine atposition 226 is designated as “Thr226Ala” or “T226A”. Multiple mutationsare separated by addition marks (“+”), e.g., “Gly205Arg+Ser411Phe” or“G205R+S411F”, representing mutations at positions 205 and 411substituting glycine (G) with arginine (R), and serine (S) withphenylalanine (F), respectively.

Deletions. For an amino acid deletion, the following nomenclature isused: Original amino acid, position*. Accordingly, the deletion ofglycine at position 195 is designated as “Gly195*” or “G195*”. Multipledeletions are separated by addition marks (“+”), e.g., “Gly195*+Ser411*”or “G195*+S411*”.

Insertions. For an amino acid insertion, the following nomenclature isused: Original amino acid, position, original amino acid, new insertedamino acid. Accordingly the insertion of lysine after glycine atposition 195 is designated “Gly195GlyLys” or “G195GK”.

Multiple modifications. Variants comprising multiple modifications areseparated by addition marks (“+”), e.g., “Arg170Tyr+Gly195Glu” or“R170Y+G195E” representing modifications at positions 170 and 195substituting tyrosine and glutamic acid for arginine and glycine,respectively.

Parent Glycoside Hydrolases

In the present invention, the parent glycoside hydrolase is (a) apolypeptide comprising an amino acid sequence which has at least 70%identity with amino acids 1 to 513 of SEQ ID NO: 2; or (b) a polypeptideencoded by a nucleotide sequence which hybridizes under at least lowstringency conditions with nucleotides 52 to 1539 of SEQ ID NO: 1, orits complementary strand.

In a first aspect, the parent glycoside hydrolase comprises an aminoacid sequence which has a degree of identity to amino acids 1 to 513 ofSEQ ID NO: 2 of at least 70%, preferably at least 75%, more preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, most preferably at least 95%, and even most preferably atleast 97%, which have glycoside hydrolase activity (hereinafter“homologous polypeptides”). For purposes of the present invention, thedegree of identity between two amino acid sequences is determined by theClustal method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENE™MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with an identity tableand the following multiple alignment parameters: Gap penalty of 10 andgap length penalty of 10. Pairwise alignment parameters were Ktuple=1,gap penalty=3, windows=5, and diagonals=5.

Substantially homologous parent glycoside hydrolases may have one ormore amino acid substitutions, deletions or additions. These changes arepreferably of a minor nature, that is conservative amino acidsubstitutions as described above and other substitutions that do notsignificantly affect the three-dimensional folding or activity of theprotein or polypeptide; small deletions, typically of one to about 30amino acids; and small amino- or carboxyl-terminal extensions, such asan amino-terminal methionine residue, a small linker peptide of up toabout 20-25 residues, or a small extension that facilitates purification(an affinity tag), such as a poly-histidine tract, or protein A (Nilssonet al., 1985, EMBO J. 4: 1075; Nilsson et al., 1991, Methods Enzymol.198: 3. See, also, in general, Ford et al., 1991, Protein Expression andPurification 2: 95-107. Examples of conservative modifications arewithin the group of basic amino acids (arginine, lysine and histidine),acidic amino acids (glutamic acid and aspartic acid), polar amino acids(glutamine and asparagine), hydrophobic amino acids (leucine, isoleucineand valine), aromatic amino acids (phenylalanine, tryptophan andtyrosine), and small amino acids (glycine, alanine, serine, threonineand methionine). Amino acid modifications, which do not generally alterthe specific activity are known in the art and are described, forexample, by H. Neurath and R. L. Hill, 1979, In, The Proteins, AcademicPress, New York. The most commonly occurring exchanges are Ala/Ser,Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly,Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, andAsp/Gly as well as the reverse (Taylor, 1986, Journal of TheoreticalBiology 119: 205-218.

Although the changes described above preferably are of a minor nature,such changes may also be of a substantive nature such as fusion oflarger polypeptides of up to 300 amino acids or more both as amino- orcarboxyl-terminal extensions.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type glycoside hydrolase. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Preferably, the parent glycoside hydrolase comprises the amino acidsequence of SEQ ID NO: 2; or an allelic variant thereof; or a fragmentthereof that has glycoside hydrolase activity. In a preferredembodiment, the parent polypeptide comprises the amino acid sequence ofSEQ ID NO: 2. In another preferred embodiment, the parent polypeptidecomprises amino acids 1 to 513 of SEQ ID NO: 2; or an allelic variantthereof; or a fragment thereof that has glycoside hydrolase activity. Inanother preferred embodiment, the parent polypeptide comprises aminoacids 1 to 513 of SEQ ID NO: 2. In another preferred embodiment, theparent polypeptide consists of the amino acid sequence of SEQ ID NO: 2;or an allelic variant thereof; or a fragment thereof that has glycosidehydrolase activity. In another preferred embodiment, the parentpolypeptide consists of the amino acid sequence of SEQ ID NO: 2. Inanother preferred embodiment, the parent polypeptide consists of aminoacids 1 to 513 of SEQ ID NO: 2 or an allelic variant thereof; or afragment thereof that has glycoside hydrolase activity. In anotherpreferred embodiment, the parent polypeptide is encoded by thenucleotide sequence contained in plasmid pAJO52 which is contained inEscherichia coli NRRL B-30683, wherein the nucleic acid sequence encodesa polypeptide having glycoside hydrolase activity. In another preferredembodiment, the parent polypeptide is encoded by the mature polypeptidecoding region contained in plasmid pAJO52 which is contained inEscherichia coli NRRL B-30683.

A fragment of SEQ ID NO: 2 is a polypeptide having one or more aminoacids deleted from the amino and/or carboxyl terminus of this amino acidsequence. Preferably, a fragment contains at least 450 amino acidresidues, more preferably at least 470 amino acid residues, and mostpreferably at least 490 amino acid residues.

In a second aspect, the parent glycoside hydrolase is encoded by anucleotide sequence which hybridizes under low stringency conditions,preferably medium stringency conditions, more preferably medium-highstringency conditions, even more preferably high stringency conditions,and most preferably very high stringency conditions with a nucleotideprobe which hybridizes under the same conditions with (i) nucleotides 52to 1539 of SEQ ID NO: 1, (ii) the genomic nucleotide sequence comprisingnucleotides 52 to 1539 of SEQ ID NO: 1, (iii) a subsequence of (i) or(ii), or (iv) a complementary strand of (i), (ii), or (iii) (J.Sambrook, E. F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). Thesubsequence of SEQ ID NO: 1 may be at least 100 contiguous nucleotidesor preferably at least 200 contiguous nucleotides. Moreover, thesubsequence may encode a polypeptide fragment which has glycosidehydrolase activity.

A subsequence of SEQ ID NO: 1, or homologue thereof, is a nucleotidesequence where one or more nucleotides have been deleted from the 5′-and/or 3′-end. Preferably, a subsequence contains at least 1350nucleotides, more preferably at least 1410 nucleotides, and mostpreferably at least 1470 nucleotides.

The parent polypeptide may also be an allelic variant of a polypeptidethat has glycoside hydrolase activity. An allelic variant denotes any oftwo or more alternative forms of a gene occupying the same chromosomallocus. Allelic variation arises naturally through mutation, and mayresult in polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

The nucleotide sequence of SEQ ID NO: 1 or a subsequence thereof, aswell as the amino acid sequence of SEQ ID NO: 2, or a fragment thereof,may be used to design nucleotide probes to identify and clone DNAencoding parent polypeptides having glycoside hydrolase activity fromstrains of different genera or species according to methods well knownin the art. In particular, such probes can be used for hybridizationwith the genomic or cDNA of the genus or species of interest, followingstandard Southern blotting procedures, in order to identify and isolatethe corresponding gene therein. Such probes can be considerably shorterthan the entire sequence, but should be at least 15, preferably at least25, and more preferably at least 35 nucleotides in length. Longer probescan also be used. Both DNA and RNA probes can be used. The probes aretypically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin).

A genomic DNA or cDNA library prepared from such other organisms may bescreened for DNA which hybridizes with the probes described above andwhich encodes a parent polypeptide having glycoside hydrolase activity.Genomic or other DNA from such other organisms may be separated byagarose or polyacrylamide gel electrophoresis, or other separationtechniques. DNA from the libraries or the separated DNA may betransferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO: 1, or a subsequence thereof, the carriermaterial is used in a Southern blot. For purposes of the presentinvention, hybridization indicates that the nucleotide sequencehybridizes to a labeled nucleotide probe corresponding to the nucleotidesequence shown in SEQ ID NO: 1, its complementary strand, or asubsequence thereof, under low to very high stringency conditions.Molecules to which the probe hybridizes can be detected using, forexample, X-ray film or any other detection means known in the art.

In a preferred embodiment, the nucleotide probe is a nucleotide sequencewhich encodes the polypeptide of SEQ ID NO: 2, or a subsequence thereof.In another preferred embodiment, the nucleotide probe is SEQ ID NO: 1.In another preferred embodiment, the nucleotide probe is nucleotides 52to 1539 of SEQ ID NO: 1. In another preferred embodiment, the nucleotideprobe is the nucleic acid sequence contained in plasmid pAJO52 which iscontained in Escherichia coli NRRL B-30683, wherein the nucleic acidsequence encodes a polypeptide having glycoside hydrolase activity. Inanother preferred embodiment, the nucleotide probe is the maturepolypeptide coding region contained in plasmid pAJO52 which is containedin Escherichia coli NRRL B-30683.

For long probes of at least 100 nucleotides in length, low to very highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmonsperm DNA, and either 25% formamide for low stringencies, 35% formamidefor medium and medium-high stringencies, or 50% formamide for high andvery high stringencies, following standard Southern blotting proceduresfor 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at least at 50° C. (low stringency), morepreferably at least at 55° C. (medium stringency), more preferably atleast at 60° C. (medium-high stringency), most preferably at least at65° C. (high stringency), and even most preferably at least at 70° C.(very high stringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(m) using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

The parent glycoside hydrolase may be obtained from microorganisms ofany genus. For purposes of the present invention, the term “obtainedfrom” as used herein in connection with a given source shall mean thatthe parent glycoside hydrolase encoded by a nucleotide sequence isproduced by the source or by a cell in which the nucleotide sequencefrom the source has been inserted. In a preferred embodiment, the parentglycoside hydrolase is secreted extracellularly.

The parent glycoside hydrolase may be a fungal glycoside hydrolase. In apreferred embodiment, the fungal glycoside hydrolase is a yeastglycoside hydrolase such as a Candida, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia glycoside hydrolase. Inanother preferred embodiment, the fungal glycoside hydrolase is afilamentous fungal glycoside hydrolase such as an Acremonium, Agaricus,Alternaria, Aspergillus, Botryosphaeria, Ceriporiopsis, Chaetomidium,Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus,Cryphonectria, Diplodia, Exidia, Fusarium, Gibberella,Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria,Magnaporthe, Melanocarpus, Meripilus, Myceliophthora, Neurospora,Penicillium, Phanerochaete, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Scytalidium, Talaromyces, Thermoascus,Thielavia, Trichoderma, Trichophaea, Verticillium, Volvariella, orXylaria glycoside hydrolase.

In a more preferred embodiment, the parent glycoside hydrolase is aSaccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomycesdiastaticus, Saccharomyces douglasii, Saccharomyces kluyveri,Saccharomyces norbensis, or Saccharomyces oviformis glycoside hydrolase.

In another more preferred embodiment, the parent glycoside hydrolase isan Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillusawamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillusfumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis,Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusariumoxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,Fusarium sarcochroum, Fusarium solani, Fusarium sporotrichioides,Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides,Fusarium venenatum, Humicola grisea, Humicola insolens, Humicolalanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum,Phanerochaete chrysosporium, Schizophyllum commune, Sclerotium rolfsii,Sporotrichum cellulophilum, Talaromyces emersonii, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride glycosidehydrolase.

In an even more preferred embodiment, the parent glycoside hydrolase isa Trichoderma reesei glycoside hydrolase, and most preferably theTrichoderma reesei cellobiohydrolase I of SEQ ID NO: 2 or the maturepolypeptide thereof. In another most preferred embodiment, the parentglycoside hydrolase is encoded by the nucleotide sequence contained inplasmid pAJO52 which is contained in Escherichia coli NRRL B-30683,wherein the nucleotide sequence encodes a polypeptide having glycosidehydrolase activity. In another most preferred embodiment, the parentglycoside hydrolase is encoded by the mature polypeptide coding regioncontained in plasmid pAJO52 which is contained in Escherichia coli NRRLB-30683.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The parent glycoside hydrolase may also be identified and obtained fromother sources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) or DNA samples obtained directly from naturalmaterials (e.g., soil, composts, water, etc,) using the above-mentionedprobes. Techniques for isolating microorganisms and DNA directly fromnatural habitats are well known in the art. The nucleotide sequenceencoding a glycoside hydrolase may then be derived by similarlyscreening a genomic or cDNA library of another microorganism or mixedDNA sample. Once a nucleotide sequence encoding a glycoside hydrolasehas been detected with suitable probe(s) as described herein, thesequence may be isolated or cloned by utilizing techniques which areknown to those of ordinary skill in the art (see, e.g., J. Sambrook, E.F. Fritsch, and T. Maniatus, 1989, Molecular Cloning, A LaboratoryManual, 2d edition, Cold Spring Harbor, N.Y.).

As defined herein, an “isolated” glycoside hydrolase is a polypeptidewhich is essentially free of other non-glycoside hydrolase polypeptides,e.g., at least 20% pure, preferably at least 40% pure, more preferablyat least 60% pure, even more preferably at least 80% pure, mostpreferably at least 90% pure, and even most preferably at least 95%pure, as determined by SDS-PAGE.

The parent glycoside hydrolase can also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator. Fusion proteins may also be constructedusing intein technology in which fusions are createdpost-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawsonet al., 1994, Science 266: 776-779).

Essential amino acids in the parent glycoside hydrolase can beidentified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, 1989, Science 244: 1081-1085). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant mutant molecules are tested for biologicalactivity (i.e., glycoside hydrolase activity) to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Left. 309:59-64. The identities of essential amino acidscan also be inferred from analysis of identities with polypeptides whichare related to a polypeptide according to the invention.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman et al., 1991, Biochem. 30:10832-10837; U.S. Pat.No. 5,223,409; WO 92/06204) and region-directed mutagenesis (Derbyshireet al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

Plasmids

The plasmid or plasmids used for preparing glycoside hydrolase variantsmay be any plasmid or vector that may be subjected to recombinant DNAprocedures. The plasmid comprising a nucleotide sequence encoding aglycoside hydrolase may be prepared by ligating the nucleotide sequenceinto a suitable plasmid, or by any other suitable method. The plasmidpreferably contains one or more selectable markers described hereinwhich permit easy selection of transformed cells. The choice of plasmidwill often depend on the host cell into which it is to be introduced.

In the present invention, the plasmid may be an autonomously replicatingplasmid, i.e., a plasmid which exists as an extrachromosomal entity, thereplication of which is distinct from chromosomal replication.

The plasmid replicator may be any plasmid replicator mediatingautonomous replication which functions in a cell. The term “plasmidreplicator” is defined herein as a sequence that enables a plasmid orvector to replicate in vivo. Examples of a plasmid replicator useful ina yeast cell are the 2 micron origin of replication, ARS1, ARS4, thecombination of ARS1 and CEN3, and the combination of ARS4 and CEN6.Examples of a plasmid replicator useful in a filamentous fungal cell areAMA1 and ANSI (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987,Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

The linearizing of the plasmid(s) can be directed toward any site withinthe plasmid. The plasmid(s) may be linearized by any suitable methodsknown in the art, for example, digestion with one or more restrictionenzymes. The linearized ends of the plasmid may be filled-in withnucleotides as described by Pompon el al., 1989, Gene 83: 15-24.However, it is preferred not to fill in the linearized ends as it mightcreate a frameshift.

To facilitate the screening process, the plasmid is preferably anexpression vector in which the nucleotide sequence in question isoperably linked to additional segments required for transcription of theDNA. In general, the expression vector is derived from a plasmid, acosmid or a bacteriophage, or may contain elements of any or all ofthese. For purposes of the present invention, the terms “plasmid” and“vector” are used interchangeably.

DNA Fragments

A library of DNA fragments to be randomly combined (or “shuffled”) withhomologous regions in the linearized plasmid(s) by in vivo recombinationmay be prepared by any suitable method. For example, the DNA fragmentmay be prepared by PCR amplification (e.g., error-prone PCR) of aplasmid comprising the nucleotide sequence, using specific primers, forexample, as described in U.S. Pat. No. 4,683,202 or Saiki et al., 1988,Science 239: 487-491. The DNA fragment may also be isolated from aplasmid comprising the desired nucleotide sequence by digestion withrestriction enzymes, followed by isolation using, for example,electrophoresis.

The DNA fragment may alternatively be prepared synthetically byestablished standard methods, e.g., the phosphoamidite method describedby Beaucage and Caruthers, 1981, Tetrahedron Letters 22: 1859-1869, orthe method described by Matthes et al., 1984, EMBO Journal 3: 801-805.According to the phosphoamidite method, oligonucleotides are synthesizedin an automatic DNA synthesizer, purified, annealed, ligated, and clonedinto suitable plasmids.

The DNA fragment may also be of mixed synthetic and genomic, mixedsynthetic and cDNA, or mixed genomic and cDNA origins prepared byligating fragments of synthetic, genomic or cDNA origin, the fragmentscorresponding to various parts of the entire nucleotide sequence, inaccordance with standard techniques.

The library of DNA fragments comprise one or more mutations of thenucleotide sequence, wherein the fragments comprise at least tworegions, one or more regions which are homologous to the 5′-region orthe 3′-region of the gap in the linearized nucleotide sequence and/orplasmid sequence and one or more second regions which are homologous tothe 5′-region or the 3′-region of the DNA fragments of the library.

The regions of the DNA fragment may be any sequence that is homologouswith the nucleotide sequence and/or plasmid sequence.

In a preferred embodiment, the regions of the DNA fragment are a5′-region and/or a 3′-region that flank a gene that encodes a glycosidehydrolase, or a 5′-region and/or a 3′-region of a gene that encodes aglycoside hydrolase.

In another preferred embodiment, the DNA fragment or fragments areprepared under conditions resulting in low, medium or high randommutagenesis frequency. To obtain low mutagenesis frequency thenucleotide sequence(s) (comprising the DNA fragment(s)) may be preparedby a standard PCR amplification method (U.S. Pat. No. 4,683,202 or Saikiet al., 1988, Science 239: 487-491). A medium or high mutagenesisfrequency may be obtained by performing the PCR amplification underconditions which reduce the fidelity of replication by a thermostablepolymerase and increase the misincorporation of nucleotides, forexample, as described by Deshler, 1992, GATA 9: 103-106; Leung et al.,1989, BioTechniques 1: 11-15.

The PCR amplification may be combined with a mutagenesis step using asuitable physical or chemical mutagenizing agent, e.g., one whichinduces transitions, transversions, inversions, scrambling, deletions,and/or insertions.

In a preferred embodiment, the DNA fragment(s) to be shuffled preferablyhave a length of about 15 bp to 8 kb, more preferably about 30 bp to 6kb, even more preferably about 40 by to 6 kb, even more preferably about80 bp to 4 kb, and most preferably about 100 bp to 2 kb, to be able tointeract optimally with the linearized plasmid.

Fungal Cells

The fungal cell, into which the mixture of plasmid/fragment nucleotidesequences are to be introduced, may be any fungal cell useful in thepresent invention. A “recombination fungal cell” is defined herein as acell capable of mediating shuffling of a number of homologous nucleotidesequences.

In a preferred embodiment, the fungal recombination cell is a yeastcell. In a more preferred embodiment, the yeast recombination cell is aCandida, Hansenula, Kluyveromyces, Pichia, Saccharomyces,Schizosaccharomyces, or Yarrowia cell.

In a most preferred embodiment, the yeast recombination cell is aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

In another preferred embodiment, the fungal recombination cell is afilamentous fungal cell. In a more preferred embodiment, the filamentousfungal recombination cell is an Acremonium, Aspergillus, Fusarium,Humicola, Mucor, Myceliophthora, Neurospora, Penicillium, Thielavia,Tolypocladium, or Trichoderma cell.

In a most preferred embodiment, the filamentous fungal recombinationcell is an Aspergillus awamori, Aspergillus foetidus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, or Aspergillusoryzae cell. In another most preferred embodiment, the filamentousfungal recombination cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred embodiment, the filamentousfungal recombination cell is a Humicola insolens, Humicola lanuginosa,Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicilliumpurpurogenum, Thielavia terrestris, Trichoderma harzianum, Trichodermakoningii, Trichoderma longibrachiatum, Trichoderma reesei, orTrichoderma viride cell.

In another most preferred embodiment, the Aspergillus cell is anAspergillus oryzae cell.

In another most preferred embodiment, the Aspergillus cell is anAspergillus niger cell.

In another most preferred embodiment, the Fusarium venenatum cell isFusarium venenatum A3/5, which was originally deposited as Fusariumgraminearum ATCC 20334 and recently reclassified as Fusarium venenatumby Yoder and Christianson, 1998, Fungal Genetics and Biology 23: 62-80and O'Donnell et al., 1998, Fungal Genetics and Biology 23: 57-67; aswell as taxonomic equivalents of Fusarium venenatum regardless of thespecies name by which they are currently known. In another mostpreferred embodiment, the Fusarium venenatum cell is a morphologicalmutant of Fusarium venenatum A3/5 or Fusarium venenatum ATCC 20334, asdisclosed in WO 97/26330.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

In Vivo Recombination

A large number of variants or homologous genes can be combined in onetransformation to efficiently create gene chimeras from the homologousgenes. The shuffling of these genes, encoding improved variants, wildtype genes, or a combination thereof, results in chimeras that can beexpressed and followed by screening to identify those chimeras with theoptimal combination of beneficial mutations. The process increasesmulti-fold the number of further improved variants that can be obtainedcompared to a process that uses only random mutagenesis (for a review,see Kuchner and Arnold, 1997, TIBTech 15: 523-530). Random mutagenesisintroduces mutations into a target nucleotide sequence, creatingdeleterious mutations much more frequently than beneficial ones. Initerative rounds of such mutagenesis, deleterious mutations accumulatemore rapidly than beneficial ones, effectively masking theidentification of beneficial mutations during screening. The randomrecombination between two or more homologous nucleotide sequences thatcontain multiple single nucleotide changes in their nucleotide sequencespotentially allows all those nucleotide changes contained in one variantto be separated from one another and to be randomly combined insteadwith any mutations present on other variants. This shuffling ofmutations provides a means by which mutations from different parentsequences can be combined with each other randomly to increase theprobability of combining nucleotide changes in a single nucleotidesequence.

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells. Mutagenized DNA molecules thatencode active polypeptides can be recovered from the host cells andrapidly sequenced using standard methods in the art. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

Efficient recombination of multiple overlapping fragments using the invivo recombination method is a means to generate chimeras from variantsor homologous genes. An overlap as small as 15 bp is sufficient forrecombination, and may be utilized for very easy domain shuffling ofeven distantly related genes. In domain shuffling, larger blocks ofnon-homologous DNA are randomly assorted by means of stretches ofhomology at their termini.

It is preferred that at least one shuffling cycle is a backcrossingcycle with the initially used DNA fragment or fragments, which may bethe wild-type DNA fragment. This eliminates non-essential mutations.Non-essential mutations may also be eliminated by using wild-type DNAfragments as the initially used input DNA material.

More than two nucleotide sequences can be shuffled at the same time, andcan be advantageous as a vast number of quite different variants can bemade rapidly without an abundance of iterative procedures. Whenrecombining many fragments from the same region, multiple overlapping ofthe fragments will increase the frequency of DNA interchange by itself,but it is also important to have a relatively high number of randomcrossovers in overlapping regions in order to recombine closely locatedvariants/differences.

An overlap as small as 15 bp between two fragments is sufficient toobtain an efficient recombination. Therefore, overlapping in the rangefrom 15 to 5000 bp, preferably from 30 bp to 500 bp, especially 30 bp to100 bp is suitable in the present invention.

In the present invention, preferably 2 or more overlapping fragments,more preferably 2 to 50 overlapping fragments, and most preferably 2 to10 overlapping fragments may advantageously be used as DNA fragments ina shuffling cycle.

Besides allowing creation of chimeric genes, employing overlappingfragments is a useful method for domain shuffling by creating smalloverlaps between DNA fragments from different domains and screening forthe best combination. For example, in the case of three DNA fragments,the overlapping regions may be as follows: the first end of the firstfragment overlaps the first end of the linearized plasmid, the first endof the second fragment overlaps the second end of the first fragment,and the second end of the second fragment overlaps the first end of thethird fragment, the first end of the third fragment overlaps (as statedabove) the second end of the second fragment, and the second end of thethird fragment overlaps the second end of the linearized plasmid.

It is understood that when using two or more DNA fragments as thestarting material, it is preferred to have continuous overlaps betweenthe ends of the plasmid and the DNA fragments.

Even though it is preferred to shuffle homologous nucleotide sequencesin the form of DNA fragment(s) and linearized plasmid(s), it is alsopossible to shuffle two or more linearized plasmids comprisinghomologous nucleotide sequences encoding polypeptides. However, in sucha case, it is important to linearize the plasmids at different sites.

In the present invention, two or more linearized plasmids and one ormore homologous DNA fragments can be used as the starting material to beshuffled. The ratio between the linearized plasmid(s) and homologous DNAfragment(s) preferably lie in the range from 20:1 to 1:50, and morepreferably from 2:1 to 1:10 (mol plasmid:mol fragments) with thespecific concentrations being from 1 pM to 10 M of the DNA.

The linearized plasmids may be gapped in such a way that the overlapbetween the fragments is deleted in the plasmid. The repair of the gapin the plasmid then requires that the fragments recombine with oneanother in addition to recombining with the ends of the gapped plasmidin order to reconstitute a circular, autonomously replicating plasmid.In a preferred embodiment, the linearization of the plasmid or vectorcreates a sufficient gap in the coding sequence of the nucleotidesequence to force the homologous recombination of the DNA fragments withthe corresponding regions of the nucleotide sequence, recreating acircular replicating plasmid.

Variants

In the present invention, the isolated variants of a parent glycosidehydrolase comprise a substitution at one or more positions correspondingto positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455,467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther comprise a substitution at one or more positions correspondingto positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255,259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO:2, wherein the variants, having glycoside hydrolase activity, compriseamino acid sequences which have a degree of identity of at least 70%,preferably at least 75%, more preferably at least 80%, more preferablyat least 85%, even more preferably at least 90%, most preferably atleast 95%, and even most preferably at least 97% to the amino acidsequence of the parent glycoside hydrolase. For purposes of the presentinvention, the degree of identity between two amino acid sequences isdetermined by the Clustal method (Higgins, 1989, CABIOS 5: 151-153)using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.)with an identity table and the following multiple alignment parameters:Gap penalty of 10 and gap length penalty of 10. Pairwise alignmentparameters were Ktuple=1, gap penalty=3, windows=5, and diagonals=5.

As defined herein, an “isolated variant” of a parent glycoside hydrolaseis a polypeptide which is essentially free of other non-glycosidehydrolase polypeptides, e.g., at least 20% pure, preferably at least 40%pure, more preferably at least 60% pure, even more preferably at least80% pure, most preferably at least 90% pure, and even most preferably atleast 95% pure, as determined by SDS-PAGE.

In a preferred embodiment, the number of amino acid substitutions in thevariants of the present invention comprise preferably 33, morepreferably 32, even more preferably 31, even more preferably 30, evenmore preferably 29, even more preferably 28, even more preferably 27,even more preferably 26, even more preferably 25, even more preferably24, even more preferably 23, even more preferably 22, even morepreferably 21, even more preferably 20, even more preferably 19, evenmore preferably 18, even more preferably 17, even more preferably 16,even more preferably 15, even more preferably 14, even more preferably13, even more preferably 12, even more preferably 11, even morepreferably 10, even more preferably 9, even more preferably 8, even morepreferably 7, even more preferably 6, even more preferably 5, even morepreferably 4, even more preferably 3, even more preferably 2, and mostpreferably 1.

In a preferred embodiment, the variant of a parent glycoside hydrolasecomprises a substitution at one or more positions corresponding topositions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467,and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally furthercomprises a substitution at one or more positions corresponding topositions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255,259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO:2. In another preferred embodiment, the variant of a parent glycosidehydrolase comprises substitutions at two or more positions correspondingto positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455,467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther comprises a substitution at one or more positions correspondingto positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255,259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO:2. In another preferred embodiment, the variant of a parent glycosidehydrolase comprises substitutions at three or more positionscorresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373,383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises a substitution at one or more positionscorresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227,246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513of SEQ ID NO: 2. In another preferred embodiment, the variant of aparent glycoside hydrolase comprises substitutions at four or morepositions corresponding to positions 21, 94, 157, 205, 206, 247, 337,350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ IDNO: 2, and optionally further comprises a substitution at one or morepositions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196,226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids1 to 513 of SEQ ID NO: 2. In another preferred embodiment, the variantof a parent glycoside hydrolase comprises substitutions at five or morepositions corresponding to positions 21, 94, 157, 205, 206, 247, 337,350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ IDNO: 2, and optionally further comprises a substitution at one or morepositions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196,226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids1 to 513 of SEQ ID NO: 2. In another preferred embodiment, the variantof a parent glycoside hydrolase comprises substitutions at six or morepositions corresponding to positions 21, 94, 157, 205, 206, 247, 337,350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ IDNO: 2, and optionally further comprises a substitution at one or morepositions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196,226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids1 to 513 of SEQ ID NO: 2. In another preferred embodiment, the variantof a parent glycoside hydrolase comprises substitutions at seven or morepositions corresponding to positions 21, 94, 157, 205, 206, 247, 337,350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ IDNO: 2, and optionally further comprises a substitution at one or morepositions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196,226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids1 to 513 of SEQ ID NO: 2. In another preferred embodiment, the variantof a parent glycoside hydrolase comprises substitutions at eight or morepositions corresponding to positions 21, 94, 157, 205, 206, 247, 337,350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ IDNO: 2, and optionally further comprises a substitution at one or morepositions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196,226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids1 to 513 of SEQ ID NO: 2. In another preferred embodiment, the variantof a parent glycoside hydrolase comprises substitutions at nine or morepositions corresponding to positions 21, 94, 157, 205, 206, 247, 337,350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ IDNO: 2, and optionally further comprises a substitution at one or morepositions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196,226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids1 to 513 of SEQ ID NO: 2. In another preferred embodiment, the variantof a parent glycoside hydrolase comprises substitutions at ten or morepositions corresponding to positions 21, 94, 157, 205, 206, 247, 337,350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ IDNO: 2, and optionally further comprises a substitution at one or morepositions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196,226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids1 to 513 of SEQ ID NO: 2. In another preferred embodiment, the variantof a parent glycoside hydrolase comprises substitutions at eleven ormore positions corresponding to positions 21, 94, 157, 205, 206, 247,337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 ofSEQ ID NO: 2, and optionally further comprises a substitution at one ormore positions corresponding to positions 8, 22, 41, 49, 57, 113, 193,196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of aminoacids 1 to 513 of SEQ ID NO: 2. In another preferred embodiment, thevariant of a parent glycoside hydrolase comprises substitutions attwelve or more positions corresponding to positions 21, 94, 157, 205,206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to513 of SEQ ID NO: 2, and optionally further comprises a substitution atone or more positions corresponding to positions 8, 22, 41, 49, 57, 113,193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 ofamino acids 1 to 513 of SEQ ID NO: 2. In another preferred embodiment,the variant of a parent glycoside hydrolase comprises substitutions atthirteen or more positions corresponding to positions 21, 94, 157, 205,206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to513 of SEQ ID NO: 2, and optionally further comprises a substitution atone or more positions corresponding to positions 8, 22, 41, 49, 57, 113,193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 ofamino acids 1 to 513 of SEQ ID NO: 2. In another preferred embodiment,the variant of a parent glycoside hydrolase comprises substitutions atpositions corresponding at least to positions 21, 94, 157, 205, 206,247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513of SEQ ID NO: 2, and optionally further comprises a substitution at oneor more positions corresponding to positions 8, 22, 41, 49, 57, 113,193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 ofamino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises a substitution ata position corresponding to position 21 of amino acids 1 to 513 of SEQID NO: 2. In another more preferred embodiment, the variant comprises asubstitution at a position corresponding to position 21 of amino acids 1to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In anothereven more preferred embodiment, the variant comprises Pro as asubstitution at a position corresponding to position 21 of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant comprises the substitution S21P of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the variant comprises a substitution ata position corresponding to position 94 of amino acids 1 to 513 of SEQID NO: 2. In another more preferred embodiment, the variant comprises asubstitution at a position corresponding to position 94 of amino acids 1to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In anothereven more preferred embodiment, the variant comprises Ser as asubstitution at a position corresponding to position 94 of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant comprises the substitution G94S of amino acids 1 to 513 of SEQID NO: 2. In another even more preferred embodiment, the variantcomprises Ala as a substitution at a position corresponding to position94 of amino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the variant comprises the substitution G94A of amino acids 1to 513 of SEQ ID NO: 2. In another even more preferred embodiment, thevariant comprises Arg as a substitution at a position corresponding toposition 94 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitution G94R ofamino acids 1 to 513 of SEQ ID NO: 2. In another even more preferredembodiment, the variant comprises Gln as a substitution at a positioncorresponding to position 94 of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitution G94Q of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises a substitution ata position corresponding to position 157 of amino acids 1 to 513 of SEQID NO: 2. In another more preferred embodiment, the variant comprises asubstitution at a position corresponding to position 157 of amino acids1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the variant comprises Arg as asubstitution at a position corresponding to position 157 of amino acids1 to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant comprises the substitution K157R of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the variant comprises a substitution ata position corresponding to position 205 of amino acids 1 to 513 of SEQID NO: 2. In another more preferred embodiment, the variant comprises asubstitution at a position corresponding to position 205 of amino acids1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the variant comprises Arg as asubstitution at a position corresponding to position 205 of amino acids1 to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant comprises the substitution G205R of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the variant comprises a substitution ata position corresponding to position 206 of amino acids 1 to 513 of SEQID NO: 2. In another more preferred embodiment, the variant comprises asubstitution at a position corresponding to position 206 of amino acids1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the variant comprises Tyr as asubstitution at a position corresponding to position 206 of amino acids1 to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant comprises the substitution H206Y of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the variant comprises a substitution ata position corresponding to position 247 of amino acids 1 to 513 of SEQID NO: 2. In another more preferred embodiment, the variant comprises asubstitution at a position corresponding to position 247 of amino acids1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the variant comprises Cys as asubstitution at a position corresponding to position 247 of amino acids1 to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant comprises the substitution Y247C of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the variant comprises a substitution ata position corresponding to position 337 of amino acids 1 to 513 of SEQID NO: 2. In another more preferred embodiment, the variant comprises asubstitution at a position corresponding to position 337 of amino acids1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the variant comprises Val as asubstitution at a position corresponding to position 337 of amino acids1 to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant comprises the substitution E337V of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the variant comprises a substitution ata position corresponding to position 350 of amino acids 1 to 513 of SEQID NO: 2. In another more preferred embodiment, the variant comprises asubstitution at a position corresponding to position 350 of amino acids1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the variant comprises Ser as asubstitution at a position corresponding to position 350 of amino acids1 to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant comprises the substitution T350S of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the variant comprises a substitution ata position corresponding to position 373 of amino acids 1 to 513 of SEQID NO: 2. In another more preferred embodiment, the variant comprises asubstitution at a position corresponding to position 373 of amino acids1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the variant comprises His as asubstitution at a position corresponding to position 373 of amino acids1 to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant comprises the substitution N373H of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the variant comprises a substitution ata position corresponding to position 383 of amino acids 1 to 513 of SEQID NO: 2. In another more preferred embodiment, the variant comprises asubstitution at a position corresponding to position 383 of amino acids1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the variant comprises Ala as asubstitution at a position corresponding to position 383 of amino acids1 to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant comprises the substitution T383A of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the variant comprises a substitution ata position corresponding to position 438 of amino acids 1 to 513 of SEQID NO: 2. In another more preferred embodiment, the variant comprises asubstitution at a position corresponding to position 438 of amino acids1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the variant comprises Leu as asubstitution at a position corresponding to position 438 of amino acids1 to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant comprises the substitution P438L of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the variant comprises a substitution ata position corresponding to position 455 of amino acids 1 to 513 of SEQID NO: 2. In another more preferred embodiment, the variant comprises asubstitution at a position corresponding to position 455 of amino acids1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the variant comprises Ala as asubstitution at a position corresponding to position 455 of amino acids1 to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant comprises the substitution T455A of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the variant comprises a substitution ata position corresponding to position 467 of amino acids 1 to 513 of SEQID NO: 2. In another more preferred embodiment, the variant comprises asubstitution at a position corresponding to position 467 of amino acids1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the variant comprises Ser as asubstitution at a position corresponding to position 467 of amino acids1 to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant comprises the substitution G467S of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the variant comprises a substitution ata position corresponding to position 486 of amino acids 1 to 513 of SEQID NO: 2. In another more preferred embodiment, the variant comprises asubstitution at a position corresponding to position 486 of amino acids1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the variant comprises Trp as asubstitution at a position corresponding to position 486 of amino acids1 to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant comprises the substitution C486W of amino acids 1 to 513 of SEQID NO: 2.

In a preferred embodiment, the variant further comprises a substitutionat a position corresponding to position 8 of amino acids 1 to 513 of SEQID NO: 2. In a more preferred embodiment, the variant further comprisesa substitution at a position corresponding to position 8 of amino acids1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In aneven more preferred embodiment, the variant further comprises Pro as asubstitution at a position corresponding to position 8 of amino acids 1to 513 of SEQ ID NO: 2. In a most preferred embodiment, the variantfurther comprises the substitution S8P of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 22 of amino acids 1to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 22 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Asp as a substitution at a position corresponding toposition 22 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionG22D of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 41 of amino acids 1to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 41 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Ile as a substitution at a position corresponding toposition 41 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionT41I of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 49 of amino acids 1to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 49 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Ser as a substitution at a position corresponding toposition 49 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionN49S of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 57 of amino acids 1to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 57 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Asn as a substitution at a position corresponding toposition 57 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionS57N of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 113 of amino acids1 to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 113 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Asn as a substitution at a position corresponding toposition 113 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionS113N of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 193 of amino acids1 to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 193 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Lys as a substitution at a position corresponding toposition 193 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionE193K of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 196 of amino acids1 to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 196 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Pro as a substitution at a position corresponding toposition 196 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionS196P of amino acids 1 to 513 of SEQ ID NO: 2. In another even morepreferred embodiment, the variant further comprises Thr as asubstitution at a position corresponding to position 196 of amino acids1 to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant further comprises the substitution S196T of amino acids 1 to 513of SEQ ID NO: 2. In another even more preferred embodiment, the variantfurther comprises Phe as a substitution at a position corresponding toposition 196 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionS196F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 226 of amino acids1 to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 226 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Ala as a substitution at a position corresponding toposition 226 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionT226A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 227 of amino acids1 to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 227 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Ala as a substitution at a position corresponding toposition 227 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionP227A of amino acids 1 to 513 of SEQ ID NO: 2. In another even morepreferred embodiment, the variant further comprises Leu as asubstitution at a position corresponding to position 227 of amino acids1 to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant further comprises the substitution P227L of amino acids 1 to 513of SEQ ID NO: 2. In another even more preferred embodiment, the variantfurther comprises Gly as a substitution at a position corresponding toposition 227 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionP227G of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 246 of amino acids1 to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 246 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Ile as a substitution at a position corresponding toposition 246 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionT246I of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 251 of amino acids1 to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 251 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Lys as a substitution at a position corresponding toposition 251 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionR251K of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 255 of amino acids1 to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 255 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Pro as a substitution at a position corresponding toposition 255 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionT255P of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 259 of amino acids1 to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 259 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Asn as a substitution at a position corresponding toposition 259 ofino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionD259N of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 301 of amino acids1 to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 301 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Ser as a substitution at a position corresponding toposition 301 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionN301S of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 356 of amino acids1 to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 356 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Ile as a substitution at a position corresponding toposition 356 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionT356I of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 371 of amino acids1 to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 371 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Cys as a substitution at a position corresponding toposition 371 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionY371C of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 411 of amino acids1 to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Phe as a substitution at a position corresponding toposition 411 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionS411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant further comprises asubstitution at a position corresponding to position 462 of amino acids1 to 513 of SEQ ID NO: 2. In another more preferred embodiment, thevariant further comprises a substitution at a position corresponding toposition 462 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the variantfurther comprises Ala as a substitution at a position corresponding toposition 462 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant further comprises the substitutionT462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 227 and 259 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 227 and 259 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Gly (or Alaor Leu) and Asn as substitutions at positions corresponding to positions227 and 259, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions P227G (or P227A or P227L)+D259N of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 227 and 486 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 227 and 486 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ala (or Leuor Gly) and Trp as substitutions at positions corresponding to positions227 and 486, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions P227A (or P227L or P227G)+C486W of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 301 and 337 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 301 and 337 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ser and Valas substitutions at positions corresponding to positions 301 and 337,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsN301S+E337V of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 196 and 350 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 196 and 350 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Pro (or Thror Phe) and Ser as substitutions at positions corresponding to positions196 and 350, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions S196P (or S196T or S196F)+T350S of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 22 and 467 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 22 and 467 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Asp and Seras substitutions at positions corresponding to positions 22 and 467,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions G22D+G467Sof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 21 and 57 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 21 and 57 of aminoacids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inan even more preferred embodiment, the variant comprises Pro and Asn assubstitutions at positions corresponding to positions 21 and 57,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions S21P+S57Nof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 205 and 411 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 205 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Arg and Pheas substitutions at positions corresponding to positions 205 and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsG205R+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 205 and 227 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 205 and 227 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Arg and Ala(or Leu or Gly) as substitutions at positions corresponding to positions205 and 227, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions G205R+P227A (or P227L or P227L) of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 205 and 206 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 205 and 206 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Arg and Tyras substitutions at positions corresponding to positions 205 and 206,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsG205R+H206Y of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 8 and 205 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 8 and 205 of aminoacids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inan even more preferred embodiment, the variant comprises Pro and Arg assubstitutions at positions corresponding to positions 8 and 205,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions S8P+G205Rof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 94 and 205 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 94 and 205 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ser (or Alaor Arg or Gln) and Arg as substitutions at positions corresponding topositions 94 and 205, respectively, of amino acids 1 to 513 of SEQ IDNO: 2. In another most preferred embodiment, the variant comprises thesubstitutions G94S (or G94A or G94R or G94Q)+G205R of amino acids 1 to513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 196 and 205 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 196 and 205 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Pro (or Thror Phe) and Arg as substitutions at positions corresponding to positions196 and 205, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions S196P (or S196T or S196F)+G205R of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 383 and 455 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 383 and 455 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ala and Alaas substitutions at positions corresponding to positions 383 and 455,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsT383A+T455A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113 and 411 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 113 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Asn and Pheas substitutions at positions corresponding to positions 113 and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsS113N+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113 and 196 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 113 and 196 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Asn and Thr(or Pro or Phe) as substitutions at positions corresponding to positions113 and 196, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions S113N+S196T (or S196P or S196F) of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113 and 462 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 113 and 462 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Asn and Alaas substitutions at positions corresponding to positions 113 and 462,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsS113N+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 196 and 462 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 196 and 462 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Thr (or Proor Phe) and Ala as substitutions at positions corresponding to positions196 and 462, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions S196T (or S196P or S196F)+T462A of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 196 and 462 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 41 and 196 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ile and Phe(or Pro or Thr) as substitutions at positions corresponding to positions41 and 196, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions T41 I+S196F (or S196P or S196T) of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 94 and 226 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 94 and 226 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ala (or Seror Arg or Gln) and Ala as substitutions at positions corresponding topositions 94 and 226, respectively, of amino acids 1 to 513 of SEQ IDNO: 2. In another most preferred embodiment, the variant comprises thesubstitutions G94A (or G94S or G94R or G94Q)+T226A of amino acids 1 to513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113, 196, and 462 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 113,196, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Asn, Thr (or Pro or Phe), and Ala as substitutions atpositions corresponding to positions 113, 196, and 462, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the variant comprises the substitutions S113N+S196T (orS196P or S196F)+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 157 and 205 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 157 and 205 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Arg and Argas substitutions at positions corresponding to positions 157 and 205,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsK157R+G205R of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 157 and 255 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 157 and 255 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Arg and Proas substitutions at positions corresponding to positions 157 and 255,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsK157R+T255P of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 205 and 255 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 205 and 255 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Arg and Proas substitutions at positions corresponding to positions 205 and 255,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsG205R+T255P of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 157, 205, and 255 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 157,205, and 255 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Arg, Arg, and Pro as substitutions at positions correspondingto positions 157, 205, and 255, respectively, of amino acids 1 to 513 ofSEQ ID NO: 2. In another most preferred embodiment, the variantcomprises the substitutions K157R+G205R+T255P of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 41 and 193 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 41 and 193 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ile and Lysas substitutions at positions corresponding to positions 41 and 193,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions T41I+E193K of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 41 and 411 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 41 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ile and Pheas substitutions at positions corresponding to positions 41 and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions T41I+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 193 and 411 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 193 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Lys and Pheas substitutions at positions corresponding to positions 193 and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsE193K+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 41, 193, and 411 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 41, 193,and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val. In an even more preferred embodiment, the variant comprisesIle, Lys, and Phe as substitutions at positions corresponding topositions 41, 193, and 411, respectively, of amino acids 1 to 513 of SEQID NO: 2. In another most preferred embodiment, the variant comprisesthe substitutions T41I+E193K+S411F of amino acids 1 to 513 of SEQ ID NO:2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 247 and 371 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 247 and 371 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Cys and Cysas substitutions at positions corresponding to positions 247 and 371,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsY247C+Y371C of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 247 and 411 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 247 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Cys and Pheas substitutions at positions corresponding to positions 247 and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsY247C+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 371 and 411 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 371 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Cys and Pheas substitutions at positions corresponding to positions 371 and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsY371C+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 247, 371, and 411 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 247,371, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Cys, Cys, and Phe as substitutions at positions correspondingto positions 247, 371, and 411, respectively, of amino acids 1 to 513 ofSEQ ID NO: 2. In another most preferred embodiment, the variantcomprises the substitutions Y247C+Y371C+S411F of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113 and 227 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 113 and 227 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Asn and Ala(or Leu or Gly) as substitutions at positions corresponding to positions113 and 227, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions S113N+P227A (or P227L or P227G) of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 196 and 227 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 196 and 227 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Thr (or Proor Phe) and Ala (or Leu or Gly) as substitutions at positionscorresponding to positions 196 and 227, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, thevariant comprises the substitutions S196T (or S196P or S196F)+P227A (orP227L or P227G) of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 227 and 462 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 227 and 462 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ala (or Leuor Gly) and Ala as substitutions at positions corresponding to positions227 and 462, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions P227A (or P227L or P227G)+T462A of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113, 196, and 227 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 142,196, and 227 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Asn, Thr (or Pro or Phe), and Ala as substitutions atpositions corresponding to positions 142, 196, and 227, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113, 227, and 462 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 113,227, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Asn, Ala (or Leu or Gly), and Ala as substitutions atpositions corresponding to positions 113, 227, and 462, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the variant comprises the substitutions S113N+P227A (orP227L or P227G)+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 196, 227, and 462 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 196,227, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Thr (or Pro or Phe), Ala (or Leu or Gly), and Ala assubstitutions at positions corresponding to positions 196, 227, and 462,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions S196T (orS196P or S196F)+P227A (or P227L or P227G)+T462A of amino acids 1 to 513of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113, 196, 227, and 462 of aminoacids 1 to 513 of SEQ ID NO: 2. In a more preferred embodiment, thevariant comprises substitutions at positions corresponding to positions113, 196, 227, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment, thevariant comprises Asn, Thr (or Pro or Phe), Ala (or Leu or Gly), and Alaas substitutions at positions corresponding to positions 113, 196, 227,and 462, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions S113N+S196T (or S196P or S196F)+P227A (or P227L orP227G)+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 49 and 113 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 49 and 113 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ser and Asnas substitutions at positions corresponding to positions 49 and 113,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions N49S+S113Nof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 49 and 227 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 49 and 227 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ser and Ala(or Leu or Gly) as substitutions at positions corresponding to positions49 and 227, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions N49S+P227A (or P227L or P227G) of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 49 and 438 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 49 and 438 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ser and Leuas substitutions at positions corresponding to positions 49 and 438,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions N49S+P438Lof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113 and 438 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 113 and 438 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Asn and Leuas substitutions at positions corresponding to positions 113 and 438,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsS113N+P438L of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 227 and 438 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 227 and 438 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ala (or Leuor Gly) and Leu as substitutions at positions corresponding to positions227 and 438, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions P227A (or P227L or P227G)+P438L of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 49, 113, and 227 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 49, 113,and 227 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val. In an even more preferred embodiment, the variant comprisesSer, Asn, and Ala (or Leu or Gly) as substitutions at positionscorresponding to positions 49, 113, and 227, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the variant comprises the substitutions N49S+S113N+P227A (or P227L orP227G) of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 49, 227, and 438 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 49, 227,and 438 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val. In an even more preferred embodiment, the variant comprisesSer, Ala (or Leu or Gly), and Leu as substitutions at positionscorresponding to positions 49, 227, and 438, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the variant comprises the substitutions N49S+P227A (or P227L orP227G)+P438L of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113, 227, and 438 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 113,227, and 438 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Asn, Ala (or Leu or Gly), and Leu as substitutions atpositions corresponding to positions 113, 227, and 438, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the variant comprises the substitutions S113N+P227A (orP227L or P227G)+P438L of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 49, 113, and 438 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 49, 113,and 438 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val. In an even more preferred embodiment, the variant comprisesSer, Asn, and Leu as substitutions at positions corresponding topositions 49, 113, and 438, respectively, of amino acids 1 to 513 of SEQID NO: 2. In another most preferred embodiment, the variant comprisesthe substitutions N49S+S113N+P438L of amino acids 1 to 513 of SEQ ID NO:2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 49, 113, 227, and 438 of aminoacids 1 to 513 of SEQ ID NO: 2. In a more preferred embodiment, thevariant comprises substitutions at positions corresponding to positions49, 113, 227, and 438 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment, thevariant comprises Ser, Asn, Ala (or Leu or Gly), and Leu assubstitutions at positions corresponding to positions 49, 113, 227, and438, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In anothermost preferred embodiment, the variant comprises the substitutionsN49S+S113N+P227A (or P227L or P227G)+P438L of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 157 and 411 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 157 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Arg and Pheas substitutions at positions corresponding to positions 157 and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsK157R+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 205 and 411 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 205 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Arg and Pheas substitutions at positions corresponding to positions 205 and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsG205R+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 255 and 411 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 255 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Pro and Pheas substitutions at positions corresponding to positions 255 and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsT255P+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 157, 255, and 411 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 157,255, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Arg, Pro, and Phe as substitutions at positions correspondingto positions 157, 255, and 411, respectively, of amino acids 1 to 513 ofSEQ ID NO: 2. In another most preferred embodiment, the variantcomprises the substitutions K157R+T255P+S411F of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 205, 255, and 411 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 205,255, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Arg, Pro, and Phe as substitutions at positions correspondingto positions 205, 255, and 411, respectively, of amino acids 1 to 513 ofSEQ ID NO: 2. In another most preferred embodiment, the variantcomprises the substitutions G205R+T255P+S411F of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 157, 205, and 411 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 157,205, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Arg, Arg, and Phe as substitutions at positions correspondingto positions 157, 205, and 411, respectively, of amino acids 1 to 513 ofSEQ ID NO: 2. In another most preferred embodiment, the variantcomprises the substitutions K157R+G205R+5411F of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 157, 205, 255, and 411 of aminoacids 1 to 513 of SEQ ID NO: 2. In a more preferred embodiment, thevariant comprises substitutions at positions corresponding to positions157, 205, 255, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment, thevariant comprises Arg, Arg, Pro, and Phe as substitutions at positionscorresponding to positions 157, 205, 255, and 411, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the variant comprises the substitutions K157R,G205R+T255P+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 196 and 411 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 196 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Thr (or Proor Phe) and Phe as substitutions at positions corresponding to positions196 and 411, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions S196T (or S196P or S196F)+S411F of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 227 and 411 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 227 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ala (or Leuor Gly) and Phe as substitutions at positions corresponding to positions227 and 411, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions P227A (or P227L or P227G)+S411F of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113, 227, and 411 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 113,227, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Asn, Ala (or Leu or Gly), and Phe as substitutions atpositions corresponding to positions 113, 227, and 411, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the variant comprises the substitutions S113N+P227A (orP227L or P227G)+5411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 196, 227, and 411 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 196,227, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Thr (or Pro or Phe), Ala (or Leu or Gly), and Phe assubstitutions at positions corresponding to positions 196, 227, and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions S196T (orS196P or S196F)+P227A (or P227L or P227G)+S411F of amino acids 1 to 513of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113, 196, and 411 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 113,196, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Asn, Thr (or Pro or Phe), and Phe as substitutions atpositions corresponding to positions 113, 196, and 411, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the variant comprises the substitutions S113N+S196T (orS196P or S196F)+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113, 196, 227, and 411 of aminoacids 1 to 513 of SEQ ID NO: 2. In a more preferred embodiment, thevariant comprises substitutions at positions corresponding to positions113, 196, 227, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment, thevariant comprises Asn, Thr (or Pro or Phe), Ala (or Leu or Gly), and Pheas substitutions at positions corresponding to positions 113, 196, 227,and 411, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions S113N+S196T (or S196P or S196F)+P227A (or P227L orP227G)+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113 and 356 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 113 and 356 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Asn and Ileas substitutions at positions corresponding to positions 113 and 356,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsS113N+T356I of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 196 and 356 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 196 and 356 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Thr (or Proor Phe) and Ile as substitutions at positions corresponding to positions196 and 356, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions S196T (or S196P or S196F)+T356I of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 227 and 356 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 227 and 356 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ala (or Leuor Gly) and Ile as substitutions at positions corresponding to positions227 and 356, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions P227A (or P227L or P227G)+T356I of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 356 and 462 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 356 and 462 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ile and Alaas substitutions at positions corresponding to positions 356 and 462,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsT356I+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113, 227, and 356 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 113,227, and 356 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Asn, Ala (or Leu or Gly), and Ile as substitutions atpositions corresponding to positions 113, 227, and 356, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the variant comprises the substitutions S113N+P227A (orP227L or P227G)+T356I of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 196, 227, and 356 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 196,227, and 356 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Thr (or Pro or Phe), Ala (or Leu or Gly), and Ile assubstitutions at positions corresponding to positions 196, 227, and 356,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions S196T (orS196P or S196F)+P227A (or P227L or P227G)+T356I of amino acids 1 to 513of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 196, 227, and 462 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 196,227, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Thr (or Pro or Phe), Ala (or Leu or Gly), and Ala assubstitutions at positions corresponding to positions 196, 227, and 462,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions S196T (orS196P or S196F)+P227A (or P227L or P227G)+T462A of amino acids 1 to 513of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 196, 356, and 462 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 196,356, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Thr (or Pro or Phe), Ile, and Ala as substitutions atpositions corresponding to positions 196, 356, and 462, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the variant comprises the substitutions S196T (or S196P orS196F)+T356I+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113, 196, and 356 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 113,196, and 356 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Asn, Thr (or Pro or Phe), and Ile as substitutions atpositions corresponding to positions 113, 196, and 356, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the variant comprises the substitutions S113N+S196T (orS196P or S196F)+T356I of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 227, 356, and 462 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 227,356, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Ala (or Leu or Gly), Ile, and Ala as substitutions atpositions corresponding to positions 227, 356, and 462, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the variant comprises the substitutions P227A (or P227L orP227G)+T356I+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113, 196, 227, and 356 of aminoacids 1 to 513 of SEQ ID NO: 2. In a more preferred embodiment, thevariant comprises substitutions at positions corresponding to positions113, 196, 227, and 356 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment, thevariant comprises Asn, Thr (or Pro or Phe), Ala (or Leu or Gly), and Ileas substitutions at positions corresponding to positions 113, 196, 227,and 356 respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions S113N+S196T (or S196P or S196F)+P227A (or P227L orP227G)+T356I of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113, 227, 356, and 462 of aminoacids 1 to 513 of SEQ ID NO: 2. In a more preferred embodiment, thevariant comprises substitutions at positions corresponding to positions113, 227, 356, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala,Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment, thevariant comprises Asn, Ala (or Leu or Gly), Ile, and Ala assubstitutions at positions corresponding to positions 113, 227, 356, and462, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In anothermost preferred embodiment, the variant comprises the substitutionsS113N+P227A (or P227L or P227G)+T356I+T462A of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113, 196, 356, and 462 of aminoacids 1 to 513 of SEQ ID NO: 2. In a more preferred embodiment, thevariant comprises substitutions at positions corresponding to positions113, 196, 356, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala,Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment, thevariant comprises Asn, Thr (or Pro or Phe), Ile, and Ala assubstitutions at positions corresponding to positions 113, 196, 356, and462, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In anothermost preferred embodiment, the variant comprises the substitutionsS113N+S196T (or S196P or S196F)+T356I+T462A of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 196, 227, 356, and 462 of aminoacids 1 to 513 of SEQ ID NO: 2. In a more preferred embodiment, thevariant comprises substitutions at positions corresponding to positions196, 227, 356, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala,Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment, thevariant comprises Thr (or Pro or Phe), Ala (or Leu or Gly), Ile, and Alaas substitutions at positions corresponding to positions 196, 227, 356,and 462, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the variant comprises thesubstitutions S196T (or S196P or S196F)+P227A (or P227L orP227G)+T356I+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 113, 196, 227, 356, and 462 ofamino acids 1 to 513 of SEQ ID NO: 2. In a more preferred embodiment,the variant comprises substitutions at positions corresponding topositions 113, 196, 227, 356, and 462 of amino acids 1 to 513 of SEQ IDNO: 2 with Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferredembodiment, the variant comprises Asn, Thr (or Pro or Phe), Ala (or Leuor Gly), Ile, and Ala as substitutions at positions corresponding topositions 113, 196, 227, 356, and 462, respectively, of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the variantcomprises the substitutions S113N+S196T (or S196P or S196F)+P227A (orP227L or P227L)+T356I+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 21 and 246 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 21 and 246 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Pro and Ileas substitutions at positions corresponding to positions 21 and 246,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions S21P+T246Iof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 21 and 251 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 21 and 251 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Pro and Lysas substitutions at positions corresponding to positions 21 and 251,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions S21P+R251Kof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 21 and 411 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 21 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Pro and Pheas substitutions at positions corresponding to positions 21 and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions S21P+S411Fof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 57 and 246 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 57 and 246 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Asn and Ileas substitutions at positions corresponding to positions 57 and 246,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions S57N+T246Iof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 57 and 251 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 57 and 251 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Asn and Lysas substitutions at positions corresponding to positions 57 and 251,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions S57N+R251Kof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 57 and 411 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 57 and 251 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Asn and Pheas substitutions at positions corresponding to positions 57 and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutions S57N+S411Fof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 246 and 251 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 246 and 251 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ile and Lysas substitutions at positions corresponding to positions 246 and 251,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsT246I+R251K of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 246 and 411 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 246 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Ile and Pheas substitutions at positions corresponding to positions 246 and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsT246I+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 251 and 411 of amino acids 1 to 513of SEQ ID NO: 2. In a more preferred embodiment, the variant comprisessubstitutions at positions corresponding to positions 251 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the variant comprises Lys and Pheas substitutions at positions corresponding to positions 251 and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsR251K+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 21, 57, and 246 of amino acids 1 to513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 21, 57,and 246 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val. In an even more preferred embodiment, the variant comprisesPro, Asn, and Ile as substitutions at positions corresponding topositions 21, 57, and 246, respectively, of amino acids 1 to 513 of SEQID NO: 2. In another most preferred embodiment, the variant comprisesthe substitutions S21P+S57N+T246I of amino acids 1 to 513 of SEQ ID NO:2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 21, 246, and 251 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 21, 246,and 251 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp,Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val. In an even more preferred embodiment, the variant comprisesPro, Ile, and Lys as substitutions at positions corresponding topositions 21, 246, and 251, respectively, of amino acids 1 to 513 of SEQID NO: 2. In another most preferred embodiment, the variant comprisesthe substitutions S21P+T246I+R251K of amino acids 1 to 513 of SEQ ID NO:2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 21, 246, and 411 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 21, 246,and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp,Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val. In an even more preferred embodiment, the variant comprisesPro, Ile, and Phe as substitutions at positions corresponding topositions 21, 246, and 411, respectively, of amino acids 1 to 513 of SEQID NO: 2. In another most preferred embodiment, the variant comprisesthe substitutions S21P+T246I+S411F of amino acids 1 to 513 of SEQ ID NO:2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 57, 246, and 251 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 57, 246,and 251 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp,Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val. In an even more preferred embodiment, the variant comprisesAsn, Ile, and Lys as substitutions at positions corresponding topositions 57, 246, and 251, respectively, of amino acids 1 to 513 of SEQID NO: 2. In another most preferred embodiment, the variant comprisesthe substitutions S57N+T246I+R251K of amino acids 1 to 513 of SEQ ID NO:2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 57, 246, and 411 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 57, 246,and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp,Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val. In an even more preferred embodiment, the variant comprisesAsn, Ile, and Phe as substitutions at positions corresponding topositions 57, 246, and 411, respectively, of amino acids 1 to 513 of SEQID NO: 2. In another most preferred embodiment, the variant comprisesthe substitutions S57N+T246I+S411F of amino acids 1 to 513 of SEQ ID NO:2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 57, 251, and 411 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 57, 251,and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp,Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val. In an even more preferred embodiment, the variant comprisesAsn, Lys, and Phe as substitutions at positions corresponding topositions 57, 251, and 411, respectively, of amino acids 1 to 513 of SEQID NO: 2. In another most preferred embodiment, the variant comprisesthe substitutions S57N+R251K+S411F of amino acids 1 to 513 of SEQ ID NO:2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 21, 57, and 251 of amino acids 1 to513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 21, 57,and 251 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro, Asp,Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp,Tyr, or Val. In an even more preferred embodiment, the variant comprisesPro, Asn, and Lys as substitutions at positions corresponding topositions 21, 57, and 251, respectively, of amino acids 1 to 513 of SEQID NO: 2. In another most preferred embodiment, the variant comprisesthe substitutions S21P+S57N+R251K of amino acids 1 to 513 of SEQ ID NO:2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 246, 251, and 411 of amino acids 1to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 246,251, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Ile, Lys, and Phe as substitutions at positions correspondingto positions 246, 251, and 411, respectively, of amino acids 1 to 513 ofSEQ ID NO: 2. In another most preferred embodiment, the variantcomprises the substitutions T246I+R251K+S411F of amino acids 1 to 513 ofSEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 21, 57, 246, and 251 of amino acids1 to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 21, 57,246, and 251 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Pro, Asn, Ile, and Lys as substitutions at positionscorresponding to positions 21, 57, 246, and 251 respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the variant comprises the substitutions S21P+S57N+T246I+R251K of aminoacids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 21, 246, 251, and 411 of aminoacids 1 to 513 of SEQ ID NO: 2. In a more preferred embodiment, thevariant comprises substitutions at positions corresponding to positions21, 246, 251, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala,Arg, Pro, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment, thevariant comprises Pro, Ile, Lys, and Phe as substitutions at positionscorresponding to positions 21, 246, 251, and 411, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the variant comprises the substitutions S21P+T246I+R251K+S411F of aminoacids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 21, 57, 251, and 411 of amino acids1 to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 21, 57,251, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Pro, Asn, Lys, and Phe as substitutions at positionscorresponding to positions 21, 57, 251, and 411, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the variant comprises the substitutions S21P+S57N+R251K+5411F of aminoacids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 57, 246, 251, and 411 of aminoacids 1 to 513 of SEQ ID NO: 2. In a more preferred embodiment, thevariant comprises substitutions at positions corresponding to positions57, 246, 251, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala,Arg, Pro, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment, thevariant comprises Asn, Ile, Lys, and Phe as substitutions at positionscorresponding to positions 57, 246, 251, and 411, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the variant comprises the substitutions S57N+T246I+R251K+S411F of aminoacids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 21, 57, 246, and 411 of amino acids1 to 513 of SEQ ID NO: 2. In a more preferred embodiment, the variantcomprises substitutions at positions corresponding to positions 21, 57,246, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Pro,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the variantcomprises Pro, Asn, Ile, and Phe as substitutions at positionscorresponding to positions 21, 57, 246, and 411, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the variant comprises the substitutions S21P+S57N+T246I+S411F of aminoacids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises substitutions atpositions corresponding to positions 21, 57, 246, 251, and 411 of aminoacids 1 to 513 of SEQ ID NO: 2. In a more preferred embodiment, thevariant comprises substitutions at positions corresponding to positions21, 57, 246, 251, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 withAla, Arg, Pro, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment,the variant comprises Pro, Asn, Ile, Lys, and Phe as substitutions atpositions corresponding to positions 21, 57, 246, 251, and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the variant comprises the substitutionsS21P+S57N+T246I+R251K+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the variant comprises at least onesubstitution selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least twosubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least threesubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least foursubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least fivesubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least sixsubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least sevensubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least eightsubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least ninesubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least tensubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least elevensubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T41I, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T3561, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least twelvesubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T41I, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T3561, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least thirteensubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T41I, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T3561, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least fourteensubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T41I, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T3561, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least fifteensubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least sixteensubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T41I, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T3561, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at leastseventeen substitutions selected from the group consisting of S21P,G94S, G94A, G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L,P227G, T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A,P438L, T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2,and optionally further comprises one or more substitutions selected fromthe group consisting of S8P, G22D, T41I, N49S, S57N, S113N, S196T,T226A, P227A, T255P, T3561, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least eighteensubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T41I, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T3561, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least nineteensubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T41I, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T3561, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises at least twentysubstitutions selected from the group consisting of S21P, G94S, G94A,G94R, G94Q, K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G,T246I, Y247C, D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L,T455A, G467S, and C486W of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further comprises one or more substitutions selected from thegroup consisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A,P227A, T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the variant comprises substitutionsconsisting of at least S21P, G94S, G94A, G94R, G94Q, K157R, E193K,S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C, D259N, R251K,N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S, and C486W ofamino acids 1 to 513 of SEQ ID NO: 2, and optionally further comprisesone or more substitutions selected from the group consisting of S8P,G22D, T41 I, N49S, S57N, S113N, S196T, T226A, P227A, T255P, T3561,Y371C, S411F, and T462A.

The variants of the present invention may further comprise one or moredeletions and/or insertions of the amino acid sequence.

In a preferred embodiment, a variant of the present invention consistsof 341 to 350, 351 to 360, 361 to 370, 371 to 380, 381 to 390, 391 to400, 401 to 410, 411 to 420, 421 to 430, 431 to 440, 441 to 450, 451 to460, 461 to 470, 471 to 480, 481 to 490, 491 to 500, or 501 to 513 aminoacids.

In another preferred embodiment, the variant comprising the substitutionT226A of amino acids 1 to 513 of SEQ ID NO: 2 is encoded by thenucleotide sequence contained in E. coli NRRL B-30657. In anotherpreferred embodiment, the variant comprising the substitutionsS113N+S196T+T462A of amino acids 1 to 513 of SEQ ID NO: 2 is encoded bythe nucleotide sequence contained in E. coli NRRL B-30658. In anotherpreferred embodiment, the variant comprising the substitutionsG22D+G467S of amino acids 1 to 513 of SEQ ID NO: 2 is encoded by thenucleotide sequence contained in E. coli NRRL B-30659. In anotherpreferred embodiment, the variant comprising the substitutions S21P+S57Nof amino acids 1 to 513 of SEQ ID NO: 2 is encoded by the nucleotidesequence contained in E. coli NRRL B-30661. In another preferredembodiment, the variant comprising the substitutions K157R+G205R+T255Pof amino acids 1 to 513 of SEQ ID NO: 2 is encoded by the nucleotidesequence contained in E. coli NRRL B-30662. In another preferredembodiment, the variant comprising the substitutions S196P+G205R ofamino acids 1 to 513 of SEQ ID NO: 2 is encoded by the nucleotidesequence contained in E. coli NRRL B-30663. In another preferredembodiment, the variant comprising the substitutionsS113N+S196T+P227A+T462A of amino acids 1 to 513 of SEQ ID NO: 2 isencoded by the nucleotide sequence contained in E. coli NRRL B-30664. Inanother preferred embodiment, the variant comprising the substitutionsT41I+E193K+S411F of amino acids 1 to 513 of SEQ ID NO: 2 is encoded bythe nucleotide sequence contained in E. coli NRRL B-30665. In anotherpreferred embodiment, the variant comprising the substitutionsN49S+S113N+P227A+P438L of amino acids 1 to 513 of SEQ ID NO: 2 isencoded by the nucleotide sequence contained in E. coli NRRL B-30666. Inanother preferred embodiment, the variant comprising the substitutionsN301 S+E337V of amino acids 1 to 513 of SEQ ID NO: 2 is encoded by thenucleotide sequence contained in E. coli NRRL B-30674. In anotherpreferred embodiment, the variant comprising the substitutionsS196P+T350S of amino acids 1 to 513 of SEQ ID NO: 2 is encoded by thenucleotide sequence contained in E. coli NRRL B-30675. In anotherpreferred embodiment, the variant comprising the substitutionsG205R+H206Y of amino acids 1 to 513 of SEQ ID NO: 2 is encoded by thenucleotide sequence contained in E. coli NRRL B-30676. In anotherpreferred embodiment, the variant comprising the substitutions S8P+G205Rof amino acids 1 to 513 of SEQ ID NO: 2 is encoded by the nucleotidesequence contained in E. coli NRRL B-30677. In another preferredembodiment, the variant comprising the substitutions G94S+G205R of aminoacids 1 to 513 of SEQ ID NO: 2 is encoded by the nucleotide sequencecontained in E. coli NRRL B-30678. In another preferred embodiment, thevariant comprising the substitutions T383A+T455A of amino acids 1 to 513of SEQ ID NO: 2 is encoded by the nucleotide sequence contained in E.coli NRRL B-30679. In another preferred embodiment, the variantcomprising the substitution N373H of amino acids 1 to 513 of SEQ ID NO:2 is encoded by the nucleotide sequence contained in E. coli NRRLB-30680. In another preferred embodiment, the variant comprising thesubstitutions Y247C+Y371C+S411F of amino acids 1 to 513 of SEQ ID NO: 2is encoded by the nucleotide sequence contained in E. coli NRRL B-30681.In another preferred embodiment, the variant comprising thesubstitutions S21P+S57N+T246I+R251K+S411F of amino acids 1 to 513 of SEQID NO: 2 is encoded by the nucleotide sequence contained in E. coli NRRLB-30682. In another preferred embodiment, the variant comprising thesubstitutions P227G+D259N of amino acids 1 to 513 of SEQ ID NO: 2 isencoded by the nucleotide sequence contained in E. coli NRRL B-30762.

The present invention also relates to methods for obtaining a variant ofa parent glycoside hydrolase, comprising: (a) introducing into theparent glycoside hydrolase a substitution at one or more positionscorresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373,383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further introducing a substitution at one or more positionscorresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227,246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513of SEQ ID NO: 2, wherein the variant has glycoside hydrolase activity;and (b) recovering the variant.

Nucleotide Sequences

The present invention also relates to isolated nucleotide sequenceswhich encode variants of a parent glycoside hydrolase, wherein thenucleotide sequences have been modified to encode the variants describedherein comprising a substitution at one or more positions correspondingto positions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455,467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther comprising a substitution at one or more positions correspondingto positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255,259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO:2.

The term “isolated nucleotide sequence” as used herein refers to anucleotide sequence which is essentially free of other nucleotidesequences, e.g., at least 20% pure, preferably at least 40% pure, morepreferably at least 60% pure, even more preferably at least 80% pure,and most preferably at least 90% pure as determined by agaroseelectrophoresis.

In a preferred embodiment, the isolated nucleotide sequence encoding thevariant comprising the substitution T226A of amino acids 1 to 513 of SEQID NO: 2 is contained in E. coli NRRL B-30657. In another preferredembodiment, the isolated nucleotide sequence encoding the variantcomprising the substitutions S113N+S196T+T462A of amino acids 1 to 513of SEQ ID NO: 2 is contained in E. coli NRRL B-30658. In anotherpreferred embodiment, the isolated nucleotide sequence encoding thevariant comprising the substitutions G22D+G467S of amino acids 1 to 513of SEQ ID NO: 2 is contained in E. coli NRRL B-30659. In anotherpreferred embodiment, the isolated nucleotide sequence encoding thevariant comprising the substitutions S21P+S57N of amino acids 1 to 513of SEQ ID NO: 2 is contained in E. coli NRRL B-30661. In anotherpreferred embodiment, the isolated nucleotide sequence encoding thevariant comprising the substitutions K157R+G205R+T255P of amino acids 1to 513 of SEQ ID NO: 2 is contained in E. coli NRRL B-30662. In anotherpreferred embodiment, the isolated nucleotide sequence encoding thevariant comprising the substitutions S196P+G205R of amino acids 1 to 513of SEQ ID NO: 2 is contained in E. coli NRRL B-30663. In anotherpreferred embodiment, the isolated nucleotide sequence encoding thevariant comprising the substitutions S113N+S196T+P227A+T462A of aminoacids 1 to 513 of SEQ ID NO: 2 is contained in E. coli NRRL B-30664. Inanother preferred embodiment, the isolated nucleotide sequence encodingthe variant comprising the substitutions T41 I+E193K+S411F of aminoacids 1 to 513 of SEQ ID NO: 2 is contained in E. coli NRRL B-30665. Inanother preferred embodiment, the isolated nucleotide sequence encodingthe variant comprising the substitutions N49S+S113N+P227A+P438L of aminoacids 1 to 513 of SEQ ID NO: 2 is contained in E. coli NRRL B-30666. Inanother preferred embodiment, the variant comprising the substitutionsN301S+E337V of amino acids 1 to 513 of SEQ ID NO: 2 is contained in E.coli NRRL B-30674. In another preferred embodiment, the isolatednucleotide sequence encoding the variant comprising the substitutionsS196P+T350S of amino acids 1 to 513 of SEQ ID NO: 2 is contained in E.coli NRRL B-30675. In another preferred embodiment, the isolatednucleotide sequence encoding the variant comprising the substitutionsG205R+H206Y of amino acids 1 to 513 of SEQ ID NO: 2 is contained in E.coli NRRL B-30676. In another preferred embodiment, the isolatednucleotide sequence encoding the variant comprising the substitutionsS8P+G205R of amino acids 1 to 513 of SEQ ID NO: 2 is contained in E.coli NRRL B-30677. In another preferred embodiment, the isolatednucleotide sequence encoding the variant comprising the substitutionsG94S+G205R of amino acids 1 to 513 of SEQ ID NO: 2 is contained in E.coli NRRL B-30678. In another preferred embodiment, the isolatednucleotide sequence encoding the variant comprising the substitutionsT383A+T455A of amino acids 1 to 513 of SEQ ID NO: 2 is contained in E.coli NRRL B-30679. In another preferred embodiment, the isolatednucleotide sequence encoding the variant comprising the substitutionN373H of amino acids 1 to 513 of SEQ ID NO: 2 is contained in E. coliNRRL B-30680. In another preferred embodiment, the isolated nucleotidesequence encoding the variant comprising the substitutionsY247C+Y371C+S411F of amino acids 1 to 513 of SEQ ID NO: 2 is containedin E. coli NRRL B-30681. In another preferred embodiment, the isolatednucleotide sequence encoding the variant comprising the substitutionsS21P+S57N+T246I+R251K+S411F of amino acids 1 to 513 of SEQ ID NO: 2 iscontained in E. coli NRRL B-30682. In another preferred embodiment, theisolated nucleotide sequence encoding the variant comprising thesubstitutions P227G+D259N of amino acids 1 to 513 of SEQ ID NO: 2 iscontained in E. coli NRRL B-30762.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga nucleotide sequence encoding a glycoside hydrolase variant of thepresent invention operably linked to one or more control sequences whichdirect the expression of the coding sequence in a suitable host cellunder conditions compatible with the control sequences. Expression willbe understood to include any step involved in the production of thevariant including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion.

“Nucleic acid construct” is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid combined and juxtaposed in a manner that would not otherwise existin nature. The term nucleic acid construct is synonymous with the termexpression cassette when the nucleic acid construct contains all thecontrol sequences required for expression of a coding sequence of avariant of the present invention. The term “coding sequence” is definedherein as a nucleotide sequence which directly specifies the amino acidsequence of its protein product. The boundaries of a genomic codingsequence are generally determined by the ATG start codon, or alternativestart codons such as GTG and TTG, located just upstream of the openreading frame at the 5′-end of the mRNA and a transcription terminatorsequence located just downstream of the open reading frame at the 3′-endof the mRNA. A coding sequence can include, but is not limited to, DNA,cDNA, and recombinant nucleotide sequences.

An isolated nucleotide sequence encoding a glycoside hydrolase variantof the present invention may be manipulated in a variety of ways toprovide for expression of the variant. Manipulation of the nucleotidesequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying nucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for the expression of a glycosidehydrolase variant of the present invention. Each control sequence may benative or foreign to the nucleotide sequence encoding the variant. Suchcontrol sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleotidesequence encoding a variant glycoside hydrolase of the presentinvention. The term “operably linked” is defined herein as aconfiguration in which a control sequence is appropriately placed at aposition relative to the coding sequence of the nucleotide sequence suchthat the control sequence directs the expression of a variant glycosidehydrolase.

The control sequence may be an appropriate promoter sequence, which isrecognized by a host cell for expression of the nucleotide sequence. Thepromoter sequence contains transcriptional control sequences whichmediate the expression of the variant glycoside hydrolase. The promotermay be any nucleic acid sequence which shows transcriptional activity inthe host cell of choice including mutant, truncated, and hybridpromoters, and may be obtained from genes encoding extracellular orintracellular polypeptides either homologous or heterologous to the hostcell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (gIaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase, Fusarium oxysporum trypsin-like protease (WO96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reeseicellobiohydrolase I, Trichoderma reesei endoglucanase I, Trichodermareesei endoglucanase II, Trichoderma reesei endoglucanase III,Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V,Trichoderma reesei xylanase I, Trichoderma reesei xylanase II,Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (ahybrid of the promoters from the genes for Aspergillus niger neutralalpha-amylase and Aspergillus oryzae triose phosphate isomerase);equivalents thereof; and mutant, truncated, and hybrid promotersthereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1,ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionine (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′-terminus of thenucleotide sequence encoding the variant glycoside hydrolase. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′-terminusof the nucleotide sequence encoding the variant glycoside hydrolase. Anyleader sequence that is functional in the host cell of choice may beused in the present invention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase. Suitable leaders for yeast host cells areobtained from the genes for Saccharomyces cerevisiae enolase (ENO-1),Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomycescerevisiae alpha-factor, and Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polypeptide-encoding sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence which is functional in the host cell of choice may be used inthe present invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of avariant glycoside hydrolase and directs the encoded polypeptide into thecell's secretory pathway. The 5′-end of the coding sequence of thenucleotide sequence may inherently contain a signal peptide codingregion naturally linked in translation reading frame with the segment ofthe coding region which encodes the secreted variant glycosidehydrolase. Alternatively, the 5′-end of the coding sequence may containa signal peptide coding region which is foreign to the coding sequence.The foreign signal peptide coding region may be required where thecoding sequence does not naturally contain a signal peptide codingregion. Alternatively, the foreign signal peptide coding region maysimply replace the natural signal peptide coding region in order toenhance secretion of the variant glycoside hydrolase. However, anysignal peptide coding region which directs the expressed polypeptideinto the secretory pathway of a host cell of choice may be used in thepresent invention.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens Ce145A cellulase, and Humicola lanuginosa lipase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of a variantglycoside hydrolase. The resultant polypeptide is known as a proenzymeor propolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Saccharomyces cerevisiae alpha-factor, Rhizomucor mieheiaspartic proteinase, and Myceliophthora thermophila laccase (WO95/33836).

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the variant glycoside hydrolase relativeto the growth of the host cell. Examples of regulatory systems are thosewhich cause the expression of the gene to be turned on or off inresponse to a chemical or physical stimulus, including the presence of aregulatory compound. In yeast, the ADH2 system or GAL1 system may beused. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillusnigerglucoamylase promoter, and Aspergillus oryzae glucoamylase promotermay be used as regulatory sequences. Other examples of regulatorysequences are those which allow for gene amplification. In eukaryoticsystems, these include the dihydrofolate reductase gene which isamplified in the presence of methotrexate, and the metallothionein geneswhich are amplified with heavy metals. In these cases, the nucleotidesequence encoding the variant glycoside hydrolase would be operablylinked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleotide sequence encoding a variant glycoside hydrolaseof the present invention, a promoter, and transcriptional andtranslational stop signals. The various nucleotide and control sequencesdescribed above may be joined together to produce a recombinantexpression vector which may include one or more convenient restrictionsites to allow for insertion or substitution of the nucleotide sequenceencoding the variant at such sites. Alternatively, the nucleotidesequence may be expressed by inserting the nucleotide sequence or anucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleotide sequence. Thechoice of the vector will typically depend on the compatibility of thevector with the host cell into which the vector is to be introduced. Thevectors may be linear or closed circular plasmids.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Suitable markers for yeast host cells are ADE2, HIS3,LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isdistinct from chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thenucleotide sequence encoding the variant or any other element of thevector for integration of the vector into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleic acid sequences for directing integration byhomologous recombination into the genome of the host cell. Theadditional nucleic acid sequences enable the vector to be integratedinto the host cell genome at a precise location(s) in the chromosome(s).To increase the likelihood of integration at a precise location, theintegrational elements should preferably contain a sufficient number ofnucleic acids, such as 100 to 10,000 base pairs, preferably 400 to10,000 base pairs, and most preferably 800 to 10,000 base pairs, whichare highly homologous with the corresponding target sequence to enhancethe probability of homologous recombination. The integrational elementsmay be any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding nucleic acid sequences. On the other hand, thevector may be integrated into the genome of the host cell bynon-homologous recombination.

For autonomous replication, the vector mayfurther comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of origins of replication for use in a yeasthost cell are the 2 micron origin of replication, ARS1, ARS4, thecombination of ARS1 and CEN3, and the combination of ARS4 and CEN6. Theorigin of replication may be one having a mutation which makesfunctioning temperature-sensitive in the host cell (see, e.g., Ehrlich,1978, Proceedings of the National Academy of Sciences USA 75: 1433).Examples of a plasmid replicator useful in a filamentous fungal cell areAMA1 and ANSI (Gems et al., 1991, Gene 98:61-67; Cullen et al., 1987,Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a nucleotide sequence of the present invention maybe inserted into the host cell to increase production of a glycosidehydrolase variant. An increase in the copy number of the nucleotidesequence can be obtained by integrating at least one additional copy ofthe sequence into the host cell genome or by including an amplifiableselectable marker gene with the nucleotide sequence where cellscontaining amplified copies of the selectable marker gene, and therebyadditional copies of the nucleotide sequence, can be selected for bycultivating the cells in the presence of the appropriate selectableagent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra) to obtain substantially pure glycoside hydrolase variants.

Host Cells

The present invention also relates to recombinant host cells, comprisinga nucleotide sequence encoding a variant glycoside hydrolase, which areadvantageously used in the recombinant production of the variant. Avector comprising a nucleotide sequence of the present invention isintroduced into a host cell so that the vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication. The choice of a host cell will to a largeextent depend upon the gene encoding the variant and its source.

The host cell may be any eukaryote, such as a mammalian, insect, plant,or fungal cell.

The host cell may be any fungal cell. “Fungi” as used herein includesthe phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (asdefined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al.,1995, supra, page 171) and all mitosporic fungi (Hawksworth et al.,1995, supra).

In a preferred embodiment, the fungal host cell is a yeast cell. “Yeast”as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In a more preferred embodiment, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred embodiment, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis cell. In another most preferred embodiment,the yeast host cell is a Kluyveromyces lactis cell. In another mostpreferred embodiment, the yeast host cell is a Yarrowia lipolytica cell.

In another preferred embodiment, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In a more preferred embodiment, the filamentous fungal host cell is, butnot limited to, an Acremonium, Aspergillus, Fusarium, Humicola, Mucor,Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, orTrichoderma cell.

In a most preferred embodiment, the filamentous fungal host cell is anAspergillus awamori, Aspergillus foetidus, Aspergillus japonicus,Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. Inanother most preferred embodiment, the filamentous fungal host cell is aFusarium bactridioides, Fusarium cerealis, Fusarium crookwellense,Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum,Fusarium trichothecioides, or Fusarium venenatum cell. In an even mostpreferred embodiment, the filamentous fungal host cell is a Fusariumvenenatum (Nirenberg sp. nov.) cell. In another most preferredembodiment, the filamentous fungal host cell is a Humicola insolens,Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila,Neurospora crassa, Penicillium purpurogenum, Thielavia terrestris,Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, or Trichoderma viride cell. Inanother even most preferred embodiment, the filamentous fungal host cellis Trichoderma reesei RutC30.

Fungal cells may be transformed according to the procedures describedherein.

Methods of Production

The present invention also relates to methods for producing a glycosidehydrolase variant, comprising:

(a) cultivating a host cell under conditions suitable for the expressionof the variant, wherein the host cell comprises a nucleotide sequencewhich has been modified to encode the variant comprising a substitutionat one or more positions corresponding to positions 21, 94, 157, 205,206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to513 of SEQ ID NO: 2, and optionally further comprising a substitution atone or more positions corresponding to positions 8, 22, 41, 49, 57, 113,193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 ofamino acids 1 to 513 of SEQ ID NO: 2, as described herein; and

(b) recovering the variant from the cultivation medium.

In the production methods of the present invention, the host cells arecultivated in a nutrient medium suitable for production of the glycosidehydrolase variant using methods known in the art. For example, the cellmay be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

In an alternative embodiment, the glycoside hydrolase variant is notrecovered, but rather a host cell of the present invention expressing avariant is used as a source of the variant.

The glycoside hydrolase variant may be detected using methods known inthe art that are specific for the polypeptides. These detection methodsmay include use of specific antibodies, formation of an enzyme product,or disappearance of an enzyme substrate. For example, an enzyme assaymay be used to determine the activity of the polypeptide as describedherein in the Examples.

The resulting glycoside hydrolase variant may be recovered by methodsknown in the art. For example, the polypeptide may be recovered from thenutrient medium by conventional procedures including, but not limitedto, collection, centrifugation, filtration, extraction, spray-drying,evaporation, or precipitation.

A glycoside hydrolase variant of the present invention may be purifiedby a variety of procedures known in the art including, but not limitedto, chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure glycosidehydrolase variants.

Other Polypeptides Having Glycoside Hydrolase Activity (1994)

The present invention also relates to isolated polypeptides havingglycoside hydrolase activity, wherein the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at one or more positionscorresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373,383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further differs at one or more positions corresponding topositions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255,259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO:2.

In a preferred embodiment, the amino acid sequence of the polypeptidediffers from amino acids 1 to 513 of SEQ ID NO: 2 by preferably 33 aminoacids, more preferably 32 amino acids, even more preferably 31 aminoacids, even more preferably 30 amino acids, even more preferably 29amino acids, even more preferably 28 amino acids, even more preferably27 amino acids, even more preferably 26 amino acids, even morepreferably 25 amino acids, even more preferably 24 amino acids, evenmore preferably 23 amino acids, even more preferably 22 amino acids,even more preferably 21 amino acids, even more preferably 20 aminoacids, even more preferably 19 amino acids, even more preferably 18amino acids, even more preferably 17 amino acids, even more preferably16 amino acids, even more preferably 15 amino acids, even morepreferably 14 amino acids, even more preferably 13 amino acids, evenmore preferably 12 amino acids, even more preferably 11 amino acids,even more preferably 10 amino acids, even more preferably 9 amino acids,even more preferably 8 amino acids, even more preferably 7 amino acids,even more preferably 6 amino acids, even more preferably 5 amino acids,even more preferably 4 amino acids, even more preferably 3 amino acids,even more preferably 2 amino acids, and most preferably 1 amino acid.

In a preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at one or more positions corresponding topositions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467,and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally furtherdiffers from SEQ ID NO: 2 at one or more positions corresponding topositions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255,259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO:2. In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at two or more positionscorresponding to positions 21, 94, 157, 205, 206, 247, 337, 350, 373,383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ ID NO: 2, andoptionally further differs from SEQ ID NO: 2 at one or more positionscorresponding to positions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227,246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids 1 to 513of SEQ ID NO: 2. In another preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 at three or morepositions corresponding to positions 21, 94, 157, 205, 206, 247, 337,350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 of SEQ IDNO: 2, and optionally further differs from SEQ ID NO: 2 at one or morepositions corresponding to positions 8, 22, 41, 49, 57, 113, 193, 196,226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of amino acids1 to 513 of SEQ ID NO: 2. In another preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 at four ormore positions corresponding to positions 21, 94, 157, 205, 206, 247,337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 ofSEQ ID NO: 2, and optionally further differs from SEQ ID NO: 2 at one ormore positions corresponding to positions 8, 22, 41, 49, 57, 113, 193,196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of aminoacids 1 to 513 of SEQ ID NO: 2. In another preferred embodiment, theamino acid sequence of the polypeptide differs from SEQ ID NO: 2 at fiveor more positions corresponding to positions 21, 94, 157, 205, 206, 247,337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 ofSEQ ID NO: 2, and optionally further differs from SEQ ID NO: 2 at one ormore positions corresponding to positions 8, 22, 41, 49, 57, 113, 193,196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of aminoacids 1 to 513 of SEQ ID NO: 2. In another preferred embodiment, theamino acid sequence of the polypeptide differs from SEQ ID NO: 2 at sixor more positions corresponding to positions 21, 94, 157, 205, 206, 247,337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513 ofSEQ ID NO: 2, and optionally further differs from SEQ ID NO: 2 at one ormore positions corresponding to positions 8, 22, 41, 49, 57, 113, 193,196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 of aminoacids 1 to 513 of SEQ ID NO: 2. In another preferred embodiment, theamino acid sequence of the polypeptide differs from SEQ ID NO: 2 atseven or more positions corresponding to positions 21, 94, 157, 205,206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to513 of SEQ ID NO: 2, and optionally further differs from SEQ ID NO: 2 atone or more positions corresponding to positions 8, 22, 41, 49, 57, 113,193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 ofamino acids 1 to 513 of SEQ ID NO: 2. In another preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 ateight or more positions corresponding to positions 21, 94, 157, 205,206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to513 of SEQ ID NO: 2, and optionally further differs from SEQ ID NO: 2 atone or more positions corresponding to positions 8, 22, 41, 49, 57, 113,193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 ofamino acids 1 to 513 of SEQ ID NO: 2. In another preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 atnine or more positions corresponding to positions 21, 94, 157, 205, 206,247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513of SEQ ID NO: 2, and optionally further differs from SEQ ID NO: 2 at oneor more positions corresponding to positions 8, 22, 41, 49, 57, 113,193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 ofamino acids 1 to 513 of SEQ ID NO: 2. In another preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 atten or more positions corresponding to positions 21, 94, 157, 205, 206,247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513of SEQ ID NO: 2, and optionally further differs from SEQ ID NO: 2 at oneor more positions corresponding to positions 8, 22, 41, 49, 57, 113,193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 ofamino acids 1 to 513 of SEQ ID NO: 2. In another preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 ateleven or more positions corresponding to positions 21, 94, 157, 205,206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to513 of SEQ ID NO: 2, and optionally further differs from SEQ ID NO: 2 atone or more positions corresponding to positions 8, 22, 41, 49, 57, 113,193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 ofamino acids 1 to 513 of SEQ ID NO: 2. In another preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 attwelve or more positions corresponding to positions 21, 94, 157, 205,206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to513 of SEQ ID NO: 2, and optionally further differs from SEQ ID NO: 2 atone or more positions corresponding to positions 8, 22, 41, 49, 57, 113,193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 ofamino acids 1 to 513 of SEQ ID NO: 2. In another preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 atthirteen or more positions corresponding to positions 21, 94, 157, 205,206, 247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to513 of SEQ ID NO: 2, and optionally further differs from SEQ ID NO: 2 atone or more positions corresponding to positions 8, 22, 41, 49, 57, 113,193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 ofamino acids 1 to 513 of SEQ ID NO: 2. In another preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 atpositions corresponding at least to positions 21, 94, 157, 205, 206,247, 337, 350, 373, 383, 438, 455, 467, and 486 of amino acids 1 to 513of SEQ ID NO: 2, and optionally further differs from SEQ ID NO: 2 at oneor more positions corresponding to positions 8, 22, 41, 49, 57, 113,193, 196, 226, 227, 246, 251, 255, 259, 301, 356, 371, 411, and 462 ofamino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at a position corresponding toposition 21 of amino acids 1 to 513 of SEQ ID NO: 2. In another morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at a position corresponding to position 21 of aminoacids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Pro at a position correspondingto position 21 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by S21P of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at a position corresponding toposition 94 of amino acids 1 to 513 of SEQ ID NO: 2.

In another more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at a position corresponding toposition 94 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 by Ser at aposition corresponding to position 94 of amino acids 1 to 513 of SEQ IDNO: 2. In another most preferred embodiment, the amino acid sequence ofthe polypeptide differs from SEQ ID NO: 2 by G94S of amino acids 1 to513 of SEQ ID NO: 2. In another even more preferred embodiment, theamino acid sequence of the polypeptide differs from SEQ ID NO: 2 by Alaat a position corresponding to position 94 of amino acids 1 to 513 ofSEQ ID NO: 2. In another most preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by G94A of aminoacids 1 to 513 of SEQ ID NO: 2. In another even more preferredembodiment, the amino acid sequence of the polypeptide differs from SEQID NO: 2 by Arg at a position corresponding to position 94 of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byG94R of amino acids 1 to 513 of SEQ ID NO: 2. In another even morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by Gln at a position corresponding to position 94 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide differs from SEQID NO: 2 by G94Q of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at a position corresponding toposition 157 of amino acids 1 to 513 of SEQ ID NO: 2. In another morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at a position corresponding to position 157 of aminoacids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Arg at a position correspondingto position 157 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by K157R of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at a position corresponding toposition 205 of amino acids 1 to 513 of SEQ ID NO: 2. In another morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at a position corresponding to position 205 of aminoacids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Arg at a position correspondingto position 205 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by G205R of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at a position corresponding toposition 206 of amino acids 1 to 513 of SEQ ID NO: 2. In another morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at a position corresponding to position 206 of aminoacids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Tyr at a position correspondingto position 206 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by H206Y of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at a position corresponding toposition 247 of amino acids 1 to 513 of SEQ ID NO: 2. In another morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at a position corresponding to position 247 of aminoacids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Cys at a position correspondingto position 247 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by Y247C of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at a position corresponding toposition 337 of amino acids 1 to 513 of SEQ ID NO: 2. In another morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at a position corresponding to position 337 of aminoacids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Val at a position correspondingto position 337 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by E337V of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at a position corresponding toposition 350 of amino acids 1 to 513 of SEQ ID NO: 2. In another morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at a position corresponding to position 350 of aminoacids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

In another even more preferred embodiment, the amino acid sequence ofthe polypeptide differs from SEQ ID NO: 2 by Ser at a positioncorresponding to position 350 of amino acids 1 to 513 of SEQ ID NO: 2.In another most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by T350S of amino acids 1 to 513of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at a position corresponding toposition 373 of amino acids 1 to 513 of SEQ ID NO: 2. In another morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at a position corresponding to position 373 of aminoacids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by His at a position correspondingto position 373 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by N373H of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at a position corresponding toposition 383 of amino acids 1 to 513 of SEQ ID NO: 2. In another morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at a position corresponding to position 383 of aminoacids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

In another even more preferred embodiment, the amino acid sequence ofthe polypeptide differs from SEQ ID NO: 2 by Ala at a positioncorresponding to position 383 of amino acids 1 to 513 of SEQ ID NO: 2.In another most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by T383A of amino acids 1 to 513of SEQ ID NO: 2. In another preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 at a positioncorresponding to position 438 of amino acids 1 to 513 of SEQ ID NO: 2.

In another more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at a position corresponding toposition 438 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In another even more preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 by Leu at aposition corresponding to position 438 of amino acids 1 to 513 of SEQ IDNO: 2. In another most preferred embodiment, the amino acid sequence ofthe polypeptide differs from SEQ ID NO: 2 by P438L of amino acids 1 to513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at a position corresponding toposition 455 of amino acids 1 to 513 of SEQ ID NO: 2. In another morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at a position corresponding to position 455 of aminoacids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ala at a position correspondingto position 455 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by T455A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at a position corresponding toposition 467 of amino acids 1 to 513 of SEQ ID NO: 2. In another morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at a position corresponding to position 467 of aminoacids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ser at a position correspondingto position 467 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by G467S of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at a position corresponding toposition 486 of amino acids 1 to 513 of SEQ ID NO: 2. In another morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at a position corresponding to position 486 of aminoacids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inanother even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Trp at a position correspondingto position 486 of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by C486W of amino acids 1 to 513 of SEQ ID NO: 2.

In a preferred embodiment, the amino acid sequence of the polypeptidefurther differs from SEQ ID NO: 2 at a position corresponding toposition 8 of amino acids 1 to 513 of SEQ ID NO: 2. In a more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 at a position corresponding to position 8 of aminoacids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln, Glu,Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. Inan even more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 by Pro at a positioncorresponding to position 8 of amino acids 1 to 513 of SEQ ID NO: 2. Ina most preferred embodiment, the amino acid sequence of the polypeptidefurther differs from SEQ ID NO: 2 by S8P of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 22 of amino acids 1 to 513 of SEQ ID NO: 2. Inanother more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 22 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Asp at a position corresponding to position 22 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by G22D of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 41 of amino acids 1 to 513 of SEQ ID NO: 2. Inanother more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 41 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Ile at a position corresponding to position 41 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by T41 I of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 49 of amino acids 1 to 513 of SEQ ID NO: 2. Inanother more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 49 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Ser at a position corresponding to position 49 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by N49S of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 57 of amino acids 1 to 513 of SEQ ID NO: 2. Inanother more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 57 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Asn at a position corresponding to position 57 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by S57N of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 113 of amino acids 1 to 513 of SEQ ID NO: 2.In another more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 113 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Asn at a position corresponding to position 113 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by S113N of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 193 of amino acids 1 to 513 of SEQ ID NO: 2.In another more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 193 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Lys at a position corresponding to position 193 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by E193K of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 196 of amino acids 1 to 513 of SEQ ID NO: 2.In another more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 196 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Pro at a position corresponding to position 196 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by S196P of amino acids 1 to 513 of SEQ ID NO: 2. Inanother even more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 by Thr at a positioncorresponding to position 196 of amino acids 1 to 513 of SEQ ID NO: 2.In another most preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 by S196T of amino acids 1to 513 of SEQ ID NO: 2. In another even more preferred embodiment, theamino acid sequence of the polypeptide further differs from SEQ ID NO: 2by Phe at a position corresponding to position 196 of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide further differs from SEQ ID NO: 2 byS196F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 226 of amino acids 1 to 513 of SEQ ID NO: 2.In another more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 226 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Ala at a position corresponding to position 226 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by T226A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 227 of amino acids 1 to 513 of SEQ ID NO: 2.In another more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 227 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Ala at a position corresponding to position 227 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by P227A of amino acids 1 to 513 of SEQ ID NO: 2. Inanother even more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 by Leu at a positioncorresponding to position 227 of amino acids 1 to 513 of SEQ ID NO: 2.In another most preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 by P227L of amino acids 1to 513 of SEQ ID NO: 2. In another even more preferred embodiment, theamino acid sequence of the polypeptide further differs from SEQ ID NO: 2by Gly at a position corresponding to position 227 of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide further differs from SEQ ID NO: 2 byP227G of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 246 of amino acids 1 to 513 of SEQ ID NO: 2.In another more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 246 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Ile at a position corresponding to position 246 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by T246I of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 251 of amino acids 1 to 513 of SEQ ID NO: 2.In another more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 251 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Lys at a position corresponding to position 251 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by R251K of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 255 of amino acids 1 to 513 of SEQ ID NO: 2.In another more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 255 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Pro at a position corresponding to position 255 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by T255P of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 259 of amino acids 1 to 513 of SEQ ID NO: 2.In another more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 259 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Asn at a position corresponding to position 259 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by D259N of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 301 of amino acids 1 to 513 of SEQ ID NO: 2.In another more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 301 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Ser at a position corresponding to position 301 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by N301 S of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 356 of amino acids 1 to 513 of SEQ ID NO: 2.In another more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 356 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Ile at a position corresponding to position 356 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by T356I of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 371 of amino acids 1 to 513 of SEQ ID NO: 2.In another more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 371 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Cys at a position corresponding to position 371 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Y371C of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 411 of amino acids 1 to 513 of SEQ ID NO: 2.In another more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 411 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Phe at a position corresponding to position 411 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 462 of amino acids 1 to 513 of SEQ ID NO: 2.In another more preferred embodiment, the amino acid sequence of thepolypeptide further differs from SEQ ID NO: 2 at a positioncorresponding to position 462 of amino acids 1 to 513 of SEQ ID NO: 2 byAla, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In another even more preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by Ala at a position corresponding to position 462 ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide further differsfrom SEQ ID NO: 2 by T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 227 and 259 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 227 and 259 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Gly (or Ala or Leu) and Asn atpositions corresponding to positions 227 and 259, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byP227G (or P227A or P227L)+D259N of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 227 and 486 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 227 and 486 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ala (or Leu or Gly) and Trp atpositions corresponding to positions 227 and 486, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byP227A (or P227L or P227G)+C486W of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 301 and 337 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 301 and 337 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ser and Val at positionscorresponding to positions 301 and 337, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byN301S+E337V of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 196 and 350 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 196 and 350 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Pro (or Thr or Phe) and Ser atpositions corresponding to positions 196 and 350, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byS196P (or S196T or S196F)+T350S of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 22 and 467 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 22 and 467 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Asp and Ser at positionscorresponding to positions 22 and 467, respectively, of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 by G22D+G467Sof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21 and 57 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 21 and 57 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Pro and Asn at positionscorresponding to positions 21 and 57, respectively, of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 by S21P+S57Nof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 205 and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 205 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Arg and Phe at positionscorresponding to positions 205 and 411, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byG205R+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 205 and 227 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 205 and 227 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Arg and Ala (or Leu or Gly) atpositions corresponding to positions 205 and 227, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byG205R+P227A (or P227L or P227G) of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 205 and 206 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 205 and 206 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Arg and Tyr at positionscorresponding to positions 205 and 206, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byG205R+H206Y of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 8 and 205 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 8 and 205 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Pro and Arg at positionscorresponding to positions 8 and 205, respectively, of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 by S8P+G205Rof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 94 and 205 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 94 and 205 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ser (or Ala or Arg or Gln) andArg at positions corresponding to positions 94 and 205, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide differs from SEQID NO: 2 by G94S (or G94A or G94R or G94Q)+G205R of amino acids 1 to 513of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 196 and 205 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 196 and 205 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Pro (or Thr or Phe) and Arg atpositions corresponding to positions 196 and 205, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byS196P (or S196T or S196F)+G205R of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 383 and 455 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 383 and 455 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ala and Ala at positionscorresponding to positions 383 and 455, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byT383A+T455A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113 and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 113 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Asn and Phe at positionscorresponding to positions 113 and 411, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byS113N+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113 and 196 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 113 and 196 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Asn and Thr (or Pro or Phe) atpositions corresponding to positions 113 and 196, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byS113N+S196T (or S196P or S196F) of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113 and 462 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 113 and 462 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Asn and Ala at positionscorresponding to positions 113 and 462, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byS113N+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 196 and 462 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 196 and 462 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Thr (or Pro or Phe) and Ala atpositions corresponding to positions 196 and 462, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byS196T (or S196P or S196F)+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 196 and 462 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 41 and 196 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ile and Phe (or Pro orThr) atpositions corresponding to positions 41 and 196, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byT41I+S196F (or S196P or S196T) of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 94 and 226 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 94 and 226 ofamino acids 1 to 513 of SEQ ID NO: 2 with Ala, Arg, Asn, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ala (or Ser or Arg or Gin) andAla at positions corresponding to positions 94 and 226, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide differs from SEQID NO: 2 by G94A (or G94S or G94R or G94Q)+T226A of amino acids 1 to 513of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 196, and 462 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 113,196, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Asn, Thr (orPro or Phe), and Ala at positions corresponding to positions 113, 196,and 462, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by S113N+S196T (or S196P orS196F)+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 157 and 205 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 157 and 205 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Arg and Arg at positionscorresponding to positions 157 and 205, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byK157R+G205R of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 157 and 255 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 157 and 255 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Arg and Pro at positionscorresponding to positions 157 and 255, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byK157R+T255P of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 205 and 255 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 205 and 255 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Arg and Pro at positionscorresponding to positions 205 and 255, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byG205R+T255P of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 157, 205, and 255 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 157,205, and 255 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Arg, Arg, andPro at positions corresponding to positions 157, 205, and 255,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by K157R+G205R+T255P of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 41 and 193 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 41 and 193 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ile and Lys at positionscorresponding to positions 41 and 193, respectively, of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 by T41I+E193K of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 41 and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 41 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ile and Phe at positionscorresponding to positions 41 and 411, respectively, of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 by T41I+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 193 and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 193 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Lys and Phe at positionscorresponding to positions 193 and 411, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byE193K+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 41, 193, and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In amore preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 41,193, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Ile, Lys, andPhe at positions corresponding to positions 41, 193, and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by T41I+E193K+S411F of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 247 and 371 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 247 and 371 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Cys and Cys at positionscorresponding to positions 247 and 371, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byY247C+Y371C of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 247 and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 247 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Cys and Phe at positionscorresponding to positions 247 and 411, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byY247C+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 371 and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 371 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Cys and Phe at positionscorresponding to positions 371 and 411, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byY371C+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 247, 371, and 411 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 247,371, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Cys, Cys, andPhe at positions corresponding to positions 247, 371, and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by Y247C+Y371C+S411F of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113 and 227 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 113 and 227 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Asn and Ala (or Leu or Gly) atpositions corresponding to positions 113 and 227, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byS113N+P227A (or P227L or P227G) of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 196 and 227 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 196 and 227 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Thr (or Pro or Phe) and Ala (orLeu or Gly) at positions corresponding to positions 196 and 227,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by S196T (or S196P or S196F)+P227A (or P227L or P227G)of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 227 and 462 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 227 and 462 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ala (or Leu or Gly) and Ala atpositions corresponding to positions 227 and 462, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byP227A (or P227L or P227G)+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 196, and 227 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 142,196, and 227 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Asn, Thr (orPro or Phe), and Ala (or Leu or Gly) at positions corresponding topositions 142, 196, and 227, respectively, of amino acids 1 to 513 ofSEQ ID NO: 2. In another most preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by S113N+S196T (orS196P or S196F)+P227A (or P227L or P227G) of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 227, and 462 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 113,227, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Asn, Ala (orLeu or Gly), and Ala at positions corresponding to positions 113, 227,and 462, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by S113N+P227A (or P227L orP227G)+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 196, 227, and 462 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 196,227, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Thr (or Pro orPhe), Ala (or Leu or Gly), and Ala at positions corresponding topositions 196, 227, and 462, respectively, of amino acids 1 to 513 ofSEQ ID NO: 2. In another most preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by S196T (or S196Por S196F)+P227A (or P227L or P227G)+T462A of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 196, and 462 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 113,196, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Asn, Thr (orPro or Phe), and Ala at positions corresponding to positions 113, 196,and 462, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by S113N+S196T (or S196P orS196F)+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 196, 227, and 462 of amino acids 1 to 513 of SEQ ID NO:2. In a more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 196, 227, and 462 of amino acids 1 to 513 of SEQ ID NO: 2by Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byAsn, Thr (or Pro or Phe), Ala (or Leu or Gly), and Ala at positionscorresponding to positions 113, 196, 227, and 462, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide differs from SEQID NO: 2 by S113N+S196T (or S196P or S196F)+P227A (or P227L orP227G)+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 49 and 113 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 49 and 113 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ser and Asn at positionscorresponding to positions 49 and 113, respectively, of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 by N49S+S113Nof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 49 and 227 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 49 and 227 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ser and Ala (or Leu or Gly) atpositions corresponding to positions 49 and 227, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byN49S+P227A (or P227L or P227G) of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 49 and 438 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 49 and 438 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ser and Leu at positionscorresponding to positions 49 and 438, respectively, of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 by N49S+P438Lof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113 and 438 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 113 and 438 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Asn and Leu at positionscorresponding to positions 113 and 438, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byS113N+P438L of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 227 and 438 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 227 and 438 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ala (or Leu or Gly) and Leu atpositions corresponding to positions 227 and 438, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byP227A (or P227L or P227G)+P438L of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 49, 113, and 227 of amino acids 1 to 513 of SEQ ID NO: 2. In amore preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 49,113, and 227 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Ser, Asn, andAla (or Leu or Gly) at positions corresponding to positions 49, 113, and227, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In anothermost preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 by N49S+S113N+P227A (or P227L or P227G) ofamino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 49, 227, and 438 of amino acids 1 to 513 of SEQ ID NO: 2. In amore preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 49,227, and 438 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Ser, Ala (orLeu or Gly), and Leu at positions corresponding to positions 49, 227,and 438, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by N49S+P227A (or P227L orP227G)+P438L of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 227, and 438 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 113,227, and 438 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Asn, Ala (orLeu or Gly), and Leu at positions corresponding to positions 113, 227,and 438, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by S113N+P227A (or P227L orP227G)+P438L of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 49, 113, and 438 of amino acids 1 to 513 of SEQ ID NO: 2. In amore preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 49,113, and 438 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Ser, Asn, andLeu at positions corresponding to positions 49, 113, and 438,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by N49S+S113N+P438L of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 49, 113, 227, and 438 of amino acids 1 to 513 of SEQ ID NO: 2.In a more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 49, 113, 227, and 438 of amino acids 1 to 513 of SEQ ID NO: 2by Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 bySer, Asn, Ala (or Leu or Gly), and Leu at positions corresponding topositions 49, 113, 227, and 438, respectively, of amino acids 1 to 513of SEQ ID NO: 2. In another most preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by N49S,S113N+P227A (or P227L or P227G)+P438L of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 157 and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 157 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Arg and Phe at positionscorresponding to positions 157 and 411, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byK157R+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 205 and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 205 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Arg and Phe at positionscorresponding to positions 205 and 411, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byG205R+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 255 and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 255 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Pro and Phe at positionscorresponding to positions 255 and 411, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byT255P+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 157, 255, and 411 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 157,255, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Arg, Pro, andPhe at positions corresponding to positions 157, 255, and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by K157R+T255P+S411F of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 205, 255, and 411 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 205,255, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Arg, Pro, andPhe at positions corresponding to positions 205, 255, and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by G205R+T255P+S411F of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 157, 205, and 411 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 157,205, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Arg, Arg, andPhe at positions corresponding to positions 157, 205, and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by K157R+G205R+S411F of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 157, 205, 255, and 411 of amino acids 1 to 513 of SEQ ID NO:2. In a more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 157, 205, 255, and 411 of amino acids 1 to 513 of SEQ ID NO: 2by Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byArg, Arg, Pro, and Phe at positions corresponding to positions 157, 205,255, and 411, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by K157R, G205R+T255P+S411F ofamino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 196 and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 196 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Thr (or Pro or Phe) and Phe atpositions corresponding to positions 196 and 411, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byS196T (or S196P or S196F)+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 227 and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 227 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ala (or Leu or Gly) and Phe atpositions corresponding to positions 227 and 411, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byP227A (or P227L or P227G)+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 227, and 411 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 113,227, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Asn, Ala (orLeu or Gly), and Phe at positions corresponding to positions 113, 227,and 411, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by S113N+P227A (or P227L orP227G)+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 196, 227, and 411 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 196,227, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Thr (or Pro orPhe), Ala (or Leu or Gly), and Phe at positions corresponding topositions 196, 227, and 411, respectively, of amino acids 1 to 513 ofSEQ ID NO: 2. In another most preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by S196T (or S196Por S196F)+P227A (or P227L or P227G)+S411F of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 196, and 411 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 113,196, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Asn, Thr (orPro or Phe), and Phe at positions corresponding to positions 113, 196,and 411, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by S113N+S196T (or S196P orS196F)+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 196, 227, and 411 of amino acids 1 to 513 of SEQ ID NO:2. In a more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 196, 227, and 411 of amino acids 1 to 513 of SEQ ID NO: 2by Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byAsn, Thr (or Pro or Phe), Ala (or Leu or Gly), and Phe at positionscorresponding to positions 113, 196, 227, and 411, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide differs from SEQID NO: 2 by S113N+S196T (or S196P or S196F)+P227A (or P227L orP227G)+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113 and 356 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 113 and 356 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Asn and Ile at positionscorresponding to positions 113 and 356, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byS113N+T356I of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 196 and 356 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 196 and 356 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Thr (or Pro or Phe) and Ile atpositions corresponding to positions 196 and 356, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byS196T (or S196P or S196F)+T356I of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 227 and 356 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 227 and 356 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ala (or Leu or Gly) and Ile atpositions corresponding to positions 227 and 356, respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byP227A (or P227L or P227G)+T356I of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 356 and 462 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 356 and 462 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ile and Ala at positionscorresponding to positions 356 and 462, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byT356I+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 227, and 356 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 113,227, and 356 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Asn, Ala (orLeu or Gly), and Ile at positions corresponding to positions 113, 227,and 356, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by S113N+P227A (or P227L orP227G)+T356I of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 196, 227, and 356 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 196,227, and 356 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Thr (or Pro orPhe), Ala (or Leu or Gly), and Ile at positions corresponding topositions 196, 227, and 356, respectively, of amino acids 1 to 513 ofSEQ ID NO: 2. In another most preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by S196T (or S196Por S196F)+P227A (or P227L or P227G)+T356I of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 196, 227, and 462 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 196,227, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Thr (or Pro orPhe), Ala (or Leu or Gly), and Ala at positions corresponding topositions 196, 227, and 462, respectively, of amino acids 1 to 513 ofSEQ ID NO: 2. In another most preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by S196T (or S196Por S196F)+P227A (or P227L or P227G)+T462A of amino acids 1 to 513 of SEQID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 196, 356, and 462 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 196,356, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Thr (or Pro orPhe), Ile, and Ala at positions corresponding to positions 196, 356, and462, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In anothermost preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 by S196T (or S196P or S196F)+T356I+T462A ofamino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 196, and 356 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 113,196, and 356 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Asn, Thr (orPro or Phe), and Ile at positions corresponding to positions 113, 196,and 356, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by S113N+S196T (or S196P orS196F)+T356I of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 227, 356, and 462 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 227,356, and 462 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Asn,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Ala (or Leu orGly), Ile, and Ala at positions corresponding to positions 227, 356, and462, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In anothermost preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 by P227A (or P227L or P227G)+T356I+T462A ofamino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 196, 227, and 356 of amino acids 1 to 513 of SEQ ID NO:2. In a more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 196, 227, and 356 of amino acids 1 to 513 of SEQ ID NO: 2by Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byAsn, Thr (or Pro or Phe), Ala (or Leu or Gly), and Ile at positionscorresponding to positions 113, 196, 227, and 356 respectively, of aminoacids 1 to 513 of SEQ ID NO: 2. In another most preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byS113N+S196T (or S196P or S196F)+P227A (or P227L or P227G)+T356I of aminoacids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 227, 356, and 462 of amino acids 1 to 513 of SEQ ID NO:2. In a more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 227, 356, and 462 of amino acids 1 to 513 of SEQ ID NO: 2by Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byAsn, Ala (or Leu or Gly), Ile, and Ala at positions corresponding topositions 113, 227, 356, and 462, respectively, of amino acids 1 to 513of SEQ ID NO: 2. In another most preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by S113N+P227A (orP227L or P227G)+T356I+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 196, 356, and 462 of amino acids 1 to 513 of SEQ ID NO:2. In a more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 196, 356, and 462 of amino acids 1 to 513 of SEQ ID NO: 2by Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byAsn, Thr (or Pro or Phe), Ile, and Ala at positions corresponding topositions 113, 196, 356, and 462, respectively, of amino acids 1 to 513of SEQ ID NO: 2. In another most preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by S113N+S196T (orS196P or S196F)+T356I+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 196, 227, 356, and 462 of amino acids 1 to 513 of SEQ ID NO:2. In a more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 196, 227, 356, and 462 of amino acids 1 to 513 of SEQ ID NO: 2by Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byThr (or Pro or Phe), Ala (or Leu or Gly), Ile, and Ala at positionscorresponding to positions 196, 227, 356, and 462, respectively, ofamino acids 1 to 513 of SEQ ID NO: 2. In another most preferredembodiment, the amino acid sequence of the polypeptide differs from SEQID NO: 2 by S196T (or S196P or S196F)+P227A (or P227L orP227G)+T356I+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 196, 227, 356, and 462 of amino acids 1 to 513 of SEQ IDNO: 2. In a more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 113, 196, 227, 356, and 462 of amino acids 1 to 513 of SEQ IDNO: 2 by Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferredembodiment, the amino acid sequence of the polypeptide differs from SEQID NO: 2 by Asn, Thr (or Pro or Phe), Ala (or Leu or Gly), Ile, and Alaat positions corresponding to positions 113, 196, 227, 356, and 462,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by S113N+S196T (or S196P or S196F)+P227A (or P227L orP227G)+T356I+T462A of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21 and 246 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 21 and 246 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Pro and Ile at positionscorresponding to positions 21 and 246, respectively, of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 by S21P+T246Iof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21 and 251 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 21 and 251 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Pro and Lys at positionscorresponding to positions 21 and 251, respectively, of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 by S21P+R251Kof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21 and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 21 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Pro and Phe at positionscorresponding to positions 21 and 411, respectively, of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 by S21P+S411Fof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 57 and 246 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 57 and 246 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Asn and Ile at positionscorresponding to positions 57 and 246, respectively, of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 by S57N+T246Iof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 57 and 251 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 57 and 251 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Asn and Lys at positionscorresponding to positions 57 and 251, respectively, of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 by S57N+R251Kof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 57 and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 57 and 251 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Asn and Phe at positionscorresponding to positions 57 and 411, respectively, of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 by S57N+S411Fof amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 246 and 251 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 246 and 251 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro, Asp, Cys, Gln,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ile and Lys at positionscorresponding to positions 246 and 251, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byT246I+R251K of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 246 and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 246 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Ile and Phe at positionscorresponding to positions 246 and 411, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byT246I+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 251 and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In a morepreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 at positions corresponding to positions 251 and 411 ofamino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro, Asp, Cys, Gin,Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.In an even more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by Lys and Phe at positionscorresponding to positions 251 and 411, respectively, of amino acids 1to 513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byR251K+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21, 57, and 246 of amino acids 1 to 513 of SEQ ID NO: 2. In amore preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 21,57, and 246 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Pro, Asn, andIle at positions corresponding to positions 21, 57, and 246,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by S21P+S57N+T246I of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21, 246, and 251 of amino acids 1 to 513 of SEQ ID NO: 2. In amore preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 21,246, and 251 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Pro, Ile, andLys at positions corresponding to positions 21, 246, and 251,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by S21P+T246I+R251K of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21, 246, and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In amore preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 21,246, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Pro, Ile, andPhe at positions corresponding to positions 21, 246, and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by S21P+T246I+S411F of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 57, 246, and 251 of amino acids 1 to 513 of SEQ ID NO: 2. In amore preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 57,246, and 251 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Asn, Ile, andLys at positions corresponding to positions 57, 246, and 251,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by S57N+T246I+R251K of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 57, 246, and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In amore preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 57,246, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro,Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Asn, Ile, andPhe at positions corresponding to positions 57, 246, and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by S57N+T246I+S411F of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 57, 251, and 411 of amino acids 1 to 513 of SEQ ID NO: 2. In amore preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 57,251, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Asn, Lys, andPhe at positions corresponding to positions 57, 251, and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by S57N+R251K+S411F of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21, 57, and 251 of amino acids 1 to 513 of SEQ ID NO: 2. In amore preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 21,57, and 251 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Pro, Asn, andLys at positions corresponding to positions 21, 57, and 251,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by S21P+S57N+R251K of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 246, 251, and 411 of amino acids 1 to 513 of SEQ ID NO: 2. Ina more preferred embodiment, the amino acid sequence of the polypeptidediffers from SEQ ID NO: 2 at positions corresponding to positions 246,251, and 411 of amino acids 1 to 513 of SEQ ID NO: 2 by Ala, Arg, Pro,Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr,Trp, Tyr, or Val. In an even more preferred embodiment, the amino acidsequence of the polypeptide differs from SEQ ID NO: 2 by Ile, Lys, andPhe at positions corresponding to positions 246, 251, and 411,respectively, of amino acids 1 to 513 of SEQ ID NO: 2. In another mostpreferred embodiment, the amino acid sequence of the polypeptide differsfrom SEQ ID NO: 2 by T246I+R251K+S411F of amino acids 1 to 513 of SEQ IDNO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21, 57, 246, and 251 of amino acids 1 to 513 of SEQ ID NO: 2.In a more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21, 57, 246, and 251 of amino acids 1 to 513 of SEQ ID NO: 2by Ala, Arg, Pro, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byPro, Asn, Ile, and Lys at positions corresponding to positions 21, 57,246, and 251 respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by S21P+S57N+T246I+R251K of aminoacids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21, 246, 251, and 411 of amino acids 1 to 513 of SEQ ID NO: 2.In a more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21, 246, 251, and 411 of amino acids 1 to 513 of SEQ ID NO: 2by Ala, Arg, Pro, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byPro, Ile, Lys, and Phe at positions corresponding to positions 21, 246,251, and 411, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by S21P+T246I+R251K+S411F of aminoacids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21, 57, 251, and 411 of amino acids 1 to 513 of SEQ ID NO: 2.In a more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21, 57, 251, and 411 of amino acids 1 to 513 of SEQ ID NO: 2by Ala, Arg, Pro, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byPro, Asn, Lys, and Phe at positions corresponding to positions 21, 57,251, and 411, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by S21P+S57N+R251K+S411F of aminoacids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 57, 246, 251, and 411 of amino acids 1 to 513 of SEQ ID NO: 2.In a more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 57, 246, 251, and 411 of amino acids 1 to 513 of SEQ ID NO: 2by Ala, Arg, Pro, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byAsn, Ile, Lys, and Phe at positions corresponding to positions 57, 246,251, and 411, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by S57N+T246I+R251K+S411F of aminoacids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21, 57, 246, and 411 of amino acids 1 to 513 of SEQ ID NO: 2.In a more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21, 57, 246, and 411 of amino acids 1 to 513 of SEQ ID NO: 2by Ala, Arg, Pro, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys, Met, Phe,Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferred embodiment,the amino acid sequence of the polypeptide differs from SEQ ID NO: 2 byPro, Asn, Ile, and Phe at positions corresponding to positions 21, 57,246, and 411, respectively, of amino acids 1 to 513 of SEQ ID NO: 2. Inanother most preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by S21P+S57N+T246I+S411F of aminoacids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21, 57, 246, 251, and 411 of amino acids 1 to 513 of SEQ IDNO: 2. In a more preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 at positions corresponding topositions 21, 57, 246, 251, and 411 of amino acids 1 to 513 of SEQ IDNO: 2 by Ala, Arg, Pro, Asp, Cys, Gin, Glu, Gly, His, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. In an even more preferredembodiment, the amino acid sequence of the polypeptide differs from SEQID NO: 2 by Pro, Asn, Ile, Lys, and Phe at positions corresponding topositions 21, 57, 246, 251, and 411, respectively, of amino acids 1 to513 of SEQ ID NO: 2. In another most preferred embodiment, the aminoacid sequence of the polypeptide differs from SEQ ID NO: 2 byS21P+S57N+T246I+R251K+S411F of amino acids 1 to 513 of SEQ ID NO: 2.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least one differenceselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, 5411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least two differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least three differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T41 I, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T3561, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least four differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least five differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least six differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least seven differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T41 I, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T3561, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least eight differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least nine differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least ten differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, 5411F, AND T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least eleven differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least twelve differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least thirteen differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least fourteen differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least fifteen differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T41I, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T3561, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least sixteen differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least seventeen differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, 5411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least eighteen differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least nineteen differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T411, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T356I, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by at least twenty differencesselected from the group consisting of S21P, G94S, G94A, G94R, G94Q,K157R, E193K, S196P, S196F, G205R, H206Y, P227L, P227G, T246I, Y247C,D259N, R251K, N301S, E337V, T350S, N373H, T383A, P438L, T455A, G467S,and C486W of amino acids 1 to 513 of SEQ ID NO: 2, and optionallyfurther differ by one or more differences selected from the groupconsisting of S8P, G22D, T41I, N49S, S57N, S113N, S196T, T226A, P227A,T255P, T3561, Y371C, S411F, and T462A.

In another preferred embodiment, the amino acid sequence of thepolypeptide differs from SEQ ID NO: 2 by the differences consisting ofat least S21P, G94S, G94A, G94R, G94Q, K157R, E193K, S196P, S196F,G205R, H206Y, P227L, P227G, T246I, Y247C, D259N, R251K, N301S, E337V,T350S, N373H, T383A, P438L, T455A, G467S, and C486W of amino acids 1 to513 of SEQ ID NO: 2, and optionally further differ by one or moredifferences selected from the group consisting of SBP, G22D, T41I, N49S,S57N, S113N, S196T, T226A, P227A, T255P, T356I, Y371C, 5411F, and T462A.

In a preferred embodiment, the polypeptide consists of 341 to 350, 351to 360, 361 to 370, 371 to 380, 381 to 390, 391 to 400, 401 to 410, 411to 420, 421 to 430, 431 to 440, 441 to 450, 451 to 460, 461 to 470, 471to 480, 481 to 490, 491 to 500, or 501 to 513 amino acids.

The isolated polypeptides have one or more improved properties comparedto the polypeptide of SEQ ID NO: 2, wherein the improved properties areselected from the group consisting of thermal activity, thermostability,pH activity, pH stability, substrate specificity, product specificity,and chemical stability, as described herein.

The present invention also relates to isolated nucleotide sequencesencoding such polypeptides, nucleic acid constructs, expression vectors,and host cells comprising the nucleotide sequences, and methods ofproducing the polypeptides having glycoside hydrolase activity,according to the same disclosure herein for glycoside hydrolasevariants.

Cellobiohydrolase I and Nucleotide Sequences Thereof

The present invention also relates to an isolated polypeptide havingcellobiohydrolase I activity and to an isolated nucleotide sequenceencoding the polypeptide.

In a preferred embodiment, the isolated polypeptide havingcellobiohydrolase I activity comprises the amino acid sequence of SEQ IDNO: 2 or an allelic variant thereof; or a fragment thereof that hascellobiohydrolase I activity. In a more preferred embodiment, thepolypeptide of the present invention comprises the amino acid sequenceof SEQ ID NO: 2. In another preferred embodiment, the polypeptide of thepresent invention comprises amino acids 1 to 513 of SEQ ID NO: 2, or anallelic variant thereof; or a fragment thereof that hascellobiohydrolase I activity. In another preferred embodiment, thepolypeptide of the present invention comprises amino acids 1 to 513 ofSEQ ID NO: 2. In another preferred embodiment, the polypeptide of thepresent invention consists of the amino acid sequence of SEQ ID NO: 2 oran allelic variant thereof; or a fragment thereof that hascellobiohydrolase I activity. In another preferred embodiment, thepolypeptide of the present invention consists of the amino acid sequenceof SEQ ID NO: 2. In another preferred embodiment, the polypeptideconsists of amino acids 1 to 513 of SEQ ID NO: 2 or an allelic variantthereof; or a fragment thereof that has cellobiohydrolase I activity. Inanother preferred embodiment, the polypeptide consists of amino acids 1to 513 of SEQ ID NO: 2.

In another preferred embodiment, the nucleotide sequence is set forth inSEQ ID NO: 1. In another more preferred embodiment, the nucleic acidsequence is the sequence contained in plasmid pAJO52 that is containedin Escherichia coli NRRL B-30683. In another preferred embodiment, thenucleotide sequence is the mature polypeptide coding region of SEQ IDNO: 1. In another more preferred embodiment, the nucleotide sequence isthe mature polypeptide coding region contained in plasmid pAJO52 that iscontained in Escherichia coli NRRL B-30683. The present invention alsoencompasses nucleotide sequences encoding a polypeptide having the aminoacid sequence of SEQ ID NO: 2, or the mature polypeptide thereof, whichdiffer from SEQ ID NO: 1 by virtue of the degeneracy of the geneticcode. The present invention also relates to subsequences of SEQ ID NO: 1which encode fragments of SEQ ID NO: 2 that have cellobiohydrolase Iactivity.

A fragment of SEQ ID NO: 2 is a polypeptide having one or more aminoacids deleted from the amino and/or carboxyl terminus of this amino acidsequence. Preferably, a fragment contains at least 450 amino acidresidues, more preferably at least 470 amino acid residues, and mostpreferably at least 490 amino acid residues.

A subsequence of SEQ ID NO:1 is a nucleic acid sequence encompassed bySEQ ID NO:1 except that one or more nucleotides from the 5′ and/or 3′end have been deleted. Preferably, a subsequence contains at least 1350nucleotides, more preferably at least 1410 nucleotides, and mostpreferably at least 1470 nucleotides.

The present invention also relates to mutant nucleic acid sequencescomprising at least one mutation in the mature polypeptide codingsequence of SEQ ID NO:1, in which the mutant nucleic acid sequenceencodes a polypeptide which consists of amino acids 1 to 513 of SEQ IDNO: 2.

The present invention also relates to nucleic acid constructs,expression vectors, and host cells comprising the nucleotide sequenceencoding the polypeptide having cellobiohydrolase activity comprisingamino acids 1 to 513 of SEQ ID NO: 2. Nucleic acid constructs,expression vectors, and host cells may be constructed as describedherein.

The present invention further relates to methods for producing thepolypeptide having cellobiohydrolase activity, comprising: (a)cultivating a strain, which in its wild-type form is capable ofproducing the polypeptide, to produce the polypeptide; and (b)recovering the polypeptide, according to the methods described herein.Preferably, the strain is of the genus Trichoderma, and more preferablyTrichoderma reesei.

The present invention also relates to methods for producing apolypeptide of the present invention comprising (a) cultivating a hostcell, comprising a nucleotide sequence encoding the polypeptide, underconditions conducive for production of the polypeptide; and (b)recovering the polypeptide, according to the methods described herein.

Degradation of Biomass to Monosaccharides, Disaccharides, andPolysaccharides

The glycoside hydrolase variants, polypeptides having glycosidehydrolase activity, and host cells of the present invention may be usedin the production of monosaccharides, disaccharides, and polysaccharidesas chemical or fermentation feedstocks from biomass for the productionof ethanol, plastics, or other products or intermediates. The glycosidevariants and polypeptides having glycoside hydrolase activity may be inthe form of a crude fermentation broth with or without the cells removedor in the form of a semi-purified or purified enzyme preparation.Alternatively, a host cell of the present invention may be used as asource of the variant or polypeptide having glycoside hydrolase activityin a fermentation process with the biomass.

Biomass can include, but is not limited to, wood resources, municipalsolid waste, wastepaper, and crop residues (see, for example, Wiselogelet al., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp.105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, BioresourceTechnology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion ofLignocellulosics, in Advances in Biochemical Engineering/Biotechnology,T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, NewYork).

The predominant polysaccharide in the primary cell wall of biomass iscellulose, the second most abundant is hemi-cellulose, and the third ispectin. The secondary cell wall, produced after the cell has stoppedgrowing, also contains polysaccharides and is strengthened throughpolymeric lignin covalently cross-linked to hemicellulose. Cellulose isa homopolymer of anhydrocellobiose and thus a linearbeta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which helps stabilize the cell wall matrix.

Three major classes of glycohydrolases are used to breakdown cellulosicbiomass:

(1) The “endo-1,4-beta-glucanases” or1,4-beta-D-glucan-4-glucanohydrolases (EC 3.2.1.4), which act randomlyon soluble and insoluble 1,4-beta-glucan substrates.

(2) The “exo-1,4-beta-D-glucanases” including both the 1,4-beta-D-glucanglucohydrolases (EC 3.2.1.74), which liberate D-glucose from1,4-beta-D-glucans and hydrolyze D-cellobiose slowly, andcellobiohydrolases (1,4-beta-D-glucan cellobiohydrolases, EC 3.2.1.91),which liberate D-cellobiose from 1,4-beta-glucans.

(3) The “beta-D-glucosidases” or beta-D-glucoside glucohydrolases (EC3.2.1.21), which act to release D-glucose units from cellobiose andsoluble cellodextrins, as well as an array of glycosides.

These three classes of enzymes work together synergistically resultingin efficient decrystallization and hydrolysis of native cellulose frombiomass to yield reducing sugars.

The glycoside hydrolase variants and polypeptides having glycosidehydrolase activity of the present invention may be used in conjunctionwith the above-noted enzymes to further degrade the cellulose componentof the biomass substrate, (see, for example, Brigham et al., 1995, inHandbook on Bioethanol (Charles E. Wyman, editor), pp. 119-141, Taylor &Francis, Washington D.C.; Lee, 1997, Journal of Biotechnology 56: 1-24).

Ethanol can be produced by enzymatic degradation of biomass andconversion of the released saccharides to ethanol. This kind of ethanolis often referred to as bioethanol or biofuel. It can be used as a fueladditive or extender in blends of from less than 1% and up to 100% (afuel substitute).

Detergent Compositions

The variants and polypeptides having glycoside hydrolase activity of thepresent invention may be added to and thus become a component of adetergent composition.

The detergent composition of the present invention may be, for example,formulated as a hand or machine laundry detergent composition includinga laundry additive composition suitable for pre-treatment of stainedfabrics and a rinse added fabric softener composition, or formulated asa detergent composition for use in general household hard surfacecleaning operations, or formulated for hand or machine dishwashingoperations.

In a specific aspect, the present invention provides a detergentadditive comprising the variants and polypeptides having glycosidehydrolase activity of the present invention. The detergent additive aswell as the detergent composition may comprise one or more other enzymessuch as a protease, lipase, cutinase, an amylase, carbohydrase,cellulase, pectinase, mannanase, arabinase, galactanase, xylanase,oxidase, e.g., a laccase, and/or peroxidase.

In general the properties of the enzymatic components should becompatible with the selected detergent, (i.e., pH-optimum, compatibilitywith other enzymatic and non-enzymatic ingredients, etc.), and theenzymatic components should be present in effective amounts.

Proteases:

Suitable proteases include those of animal, vegetable or microbialorigin. Microbial origin is preferred. Chemically modified or proteinengineered mutants are included. The protease may be a serine proteaseor a metalloprotease, preferably an alkaline microbial protease or atrypsin-like protease. Examples of alkaline proteases are subtilisins,especially those derived from Bacillus, e.g., subtilisin Novo,subtilisin Carlsberg, subtilisin 309, subtilisin 147 and subtilisin 168(described in WO 89/06279). Examples of trypsin-like proteases aretrypsin (e.g., of porcine or bovine origin) and the Fusarium proteasedescribed in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and274.

Preferred commercially available protease enzymes include Alcalase™,Savinase™ Primase™, Duralase™, Esperase™, and Kannase™ (Novozymes A/S),Maxatase™, Maxacal™ Maxapem™, Properase™, Purafect™, Purafect OxP™,FN2™, and FN3™ (Genencor International Inc.).

Lipases:

Suitable lipases include those of bacterial or fungal origin. Chemicallymodified or protein engineered mutants are included. Examples of usefullipases include lipases from Humicola (synonym Thermomyces), e.g., fromH. lanuginosa (T. lanuginosus) as described in EP 258 068 and EP 305 216or from H. insolens as described in WO 96/13580, a Pseudomonas lipase,e.g., from P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P.cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens,Pseudomonas sp. strain SD 705 (WO 95/06720 and WO 96/27002), P.wisconsinensis (WO 96/12012), a Bacillus lipase, e.g., from B. subtilis(Dartois et al., 1993, Biochemica et Biophysica Acta, 1131, 253-360), B.stearothermophilus (JP 64/744992) or B. pumilus (WO 91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipases include Lipolase™ and LipolaseUltra™ (Novozymes A/S).

Amylases:

Suitable amylases (α and/or β) include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Amylases include, for example, a-amylases obtained from Bacillus, e.g.,a special strain of Bacillus licheniformis, described in more detail inGB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from GenencorInternational Inc.).

Cellulases:

Suitable cellulases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Suitablecellulases include cellulases from the genera Bacillus, Pseudomonas,Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulasesproduced from Humicola insolens, Myceliophthora thermophila and Fusariumoxysporum disclosed in U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,648,263,U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving colour care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. No. 5,457,046, U.S. Pat. No.5,686,593, U.S. Pat. No. 5,763,254, WO 95/24471, WO 98/12307 andPCT/DK98/00299.

Commercially available cellulases include Celluzyme™, and Carezyme™(Novozymes A/S), Clazinase™, and Puradax HA™ (Genencor InternationalInc.), and KAC-500(B)™ (Kao Corporation).

Peroxidases/Oxidases:

Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically modified or protein engineered mutants areincluded. Examples of useful peroxidases include peroxidases fromCoprinus, e.g., from C. cinereus, and variants thereof as thosedescribed in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The enzymatic component(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the present invention, i.e., a separate additive ora combinedadditive, can be formulated, for example, as a granulate, liquid,slurry, etc. Preferred detergent additive formulations are granulates,in particular non-dusting granulates, liquids, in particular stabilizedliquids, or slurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the present invention may be in anyconvenient form, e.g., a bar, a tablet, a powder, a granule, a paste ora liquid. A liquid detergent may be aqueous, typically containing up to70% water and 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers,and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system that may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.

The enzymatic component(s) of the detergent composition of the presentinvention may be stabilized using conventional stabilizing agents, e.g.,a polyol such as propylene glycol or glycerol, a sugar or sugar alcohol,lactic acid, boric acid, ora boric acid derivative, e.g., an aromaticborate ester, or a phenyl boronic acid derivative such as 4-formylphenylboronic acid, and the composition may be formulated as described in, forexample, WO 92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

In the detergent compositions any enzymatic component, in particular thevariants and polypeptides having glycoside hydrolase activity of thepresent invention, may be added in an amount corresponding to 0.01-100mg of enzyme protein per liter of wash liquor, preferably 0.05-5 mg ofenzyme protein per liter of wash liquor, in particular 0.1-1 mg ofenzyme protein per liter of wash liquor.

The variants and polypeptides having glycoside hydrolase activity of thepresent invention may additionally be incorporated in the detergentformulations disclosed in WO 97/07202 which is hereby incorporated asreference.

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with a nucleotide sequenceencoding a variant or polypeptides having glycoside hydrolase activityof the present invention so as to express and produce the variant orpolypeptide in recoverable quantities. The variant or polypeptide may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the variant or polypeptide may be used as such forimproving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a variant or polypeptideof the present invention may be constructed in accordance with methodsknown in the art. In short, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a variant orpolypeptide of the present invention into the plant host genome andpropagating the resulting modified plant or plant cell into a transgenicplant or plant cell.

Conveniently, the expression construct is a nucleic acid construct whichcomprises a nucleic acid sequence encoding a variant or polypeptide ofthe present invention operably linked with appropriate regulatorysequences required for expression of the nucleic acid sequence in theplant or plant part of choice. Furthermore, the expression construct maycomprise a selectable marker useful for identifying host cells intowhich the expression construct has been integrated and DNA sequencesnecessary for introduction of the construct into the plant in question(the latter depends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the variant or polypeptideis desired to be expressed. For example, the expression of the geneencoding a variant or polypeptide of the present invention may beconstitutive or inducible, or may be developmental, stage or tissuespecific, and the gene product may be targeted to a specific tissue orplant part such as seeds or leaves. Regulatory sequences are, forexample, described by Tague et al., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294, Christensen et al., 1992, Plant Mo. Biol. 18: 675-689; Zhang etal., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forexample, the promoter enhancer element may be an intron which is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. Xu et al., 1993, supra, disclose the use ofthe first intron of the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods for producing a variant orpolypeptide of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a nucleic acid sequenceencoding a variant or polypeptide having glycoside hydrolase activity ofthe present invention under conditions conducive for production of thevariant; and (b) recovering the variant or polypeptide.

Other Uses

The glycoside hydrolase variants or polypeptides having glycosidehydrolase activity of the present invention may also be used in thetreatment of textiles as biopolishing agents and for reducing fuzz,pilling, texture modification, and stonewashing (N. K. Lange, in P.Suominen, T. Reinikainen (Eds.), Trichoderma reesei Cellulases and OtherHydrolases, Foundation for Biotechnical and Industrial Fermentationresearch, Helsinki, 1993, pp. 263-272). In addition, the describedvariants or polypeptides having glycoside hydrolase activity may also beused in wood processing for biopulping or debarking, paper manufacturingfor fiber modification, bleaching, and reduction of refining energycosts, whitewater treatment, important to wastewater recycling,lignocellulosic fiber recycling such as deinking and secondary fiberprocessing, and wood residue utilization (S. D, Mansfield and A. R.Esteghlalian in S. D, Mansfield and J. N. Saddler (Eds.), Applicationsof Enzymes to Lignocellulosics, ACS Symposium Series 855, Washington,D.C., 2003, pp. 2-29).

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES Strains

Trichoderma reesei RutC30 (ATCC 56765; Montenecourt and Eveleigh, 1979,Adv. Chem. Ser. 181: 289-301) was derived from Trichoderma reesei Qm6A(ATCC 13631; Mandels and Reese, 1957, J. Bacteriol. 73: 269-278).

Aspergillus oryzae Ja1250 strain (WO 99/61651) was used for expressionof the thermostable beta-glucosidase.

Saccharomyces cerevisiae YNG 344 (MATα, ura3-52, leu-2Δ2, pep4Δ1,his4-539, cir⁰) was used to generate libraries of mutagenized glycosidehydrolase (CeI7A).

Bacterial strains used to generate plasmids were Epicurian coli XL-10Gold ultracompetent cells, (Stratagene, Inc., La Jolla, Calif.).

Media and Solutions

Yeast selection medium was composed per liter of 6.7 g of yeast nitrogenbase, 0.8 g of complete supplement mixture (CSM-URA, Qbiogene, Inc.,Carlsbad, Calif.; lacking uracil and containing 40 mg/ml of adenine), 5g of casamino acid, and 20 g of Noble agar. The medium also contained 50mM succinate pH 5.0, 2% glucose, and 25 mg of chloramphenicol per ml.

Yeast screening medium was composed per liter of 6.7 g of yeast nitrogenbase, 0.8 g of CSM-URA, 5 g of casamino acid, and 20 g of Noble agar.The medium also contained 50 mM succinate pH 5.0, 2% galactose, 0.1%glucose, and 25 mg of chloramphenicol per ml.

YPD medium was composed per liter of 10 g of yeast extract, 20 g ofbacto peptone, and 40 ml of 50% glucose.

Cellulase-inducing media was composed per liter of 20 g of ArbocelB800-natural cellulose fibers (J. Rettenmaier USA LP, Schoolcraft,Michigan), 10 g of corn steep solids (Sigma Chemical Co., St. Louis,Mo.), 1.45 g of (NH₄)₂SO₄, 2.08 g of KH₂PO₄, 0.28 g of CaCl₂, 0.42 g ofMgSO₄.7H₂O, 0.42 ml of Trichoderma reesei trace metals solution, and 2drops of pluronic acid; pH to 6.0 with 10 N NaoH.

Trichoderma reesei trace metals solution was composed per liter of 216 gof FeCl₃.6H₂O, 58 g of ZnSO₄.7H₂O, 27 g of MnSO₄—H₂O, 10 g ofCuSO₄.5H₂O, 2.4 g of H₃BO₃, and 336 g of citric acid.

COVE selection plates were composed per liter of 342.3 g of sucrose, 20ml of COVE salt solution, 10 mM acetamide, 15 mM CsCl₂, and 25 g ofNoble agar.

COVE2 plus uridine plates were composed per liter of 30 g of sucrose, 20ml COVE salt solution, 10 mM acetamide, 10 mM uridine, and 25 g of Nobleagar.

COVE salt solution was composed per liter of 26 g of KCl, 26 g ofMgSO₄.7H₂O, 76 g of KH₂PO₄, and 50 ml of COVE trace metals.

COVE trace metals solution was composed per liter of 0.04 g ofNaB₄O₇.10H₂O, 0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g ofMnSO₄—H₂O, 0.8 g of Na₂MoO₂.2H₂O, and 10 g of ZnSO₄.7H₂O.

PDA medium was composed per liter of 39 g potato dextrose agar.

1×SSC was composed per liter of 8.765 g sodium chloride and 4.41 gsodium citrate.

PEG Buffer was composed per liter of 500 g of PEG 4000 (BDH, Poole,England), 10 mM CaCl₂, and 10 mM Tris-HCl pH 7.5 (filter sterilized).

STC was composed per liter of 1 M sorbitol, 10 mM CaCl₂, and 10 mMTris-HCl pH 7.5, and was filter sterilized.

Trichoderma reesei Inoculum Medium was composed per liter of 20 g ofglucose, 10 g of corn steep solids (Sigma Chemical Co., St. Louis, Mo.),1.45 g of (NH₄)₂SO₄, 2.08 g of KH₂PO₄, 0.28 g of CaCl₂, 0.42 g ofMgSO₄.7H₂O, 0.42 ml of Trichoderma reesei trace metals solution, and 2drops of pluronic acid; final pH 5.0.

Trichoderma reesei Fermentation Medium was composed per liter of 4 g ofglucose, 10 g of corn steep solids, 30 g of Arbocel B800-naturalcellulose fibers (J. Rettenmaier USA LP, Schoolcraft, Michigan), 3.8 gof (NH₄)₂SO₄, 2.8 g of KH₂PO₄, 2.08 g of CaCl₂, 1.63 g of MgSO₄.7H₂O,0.75 ml of Trichoderma reesei trace metals solution, and 1.8 ml ofpluronic acid.

Trichoderma reesei Feed Medium was composed per liter of 600 g ofglucose, 20 g of Cellulose B800, 35.5 g of H₃PO₄, and 5 ml of pluronicacid.

Aspergillus oryzae Inoculum Medium was composed per liter of 50 g ofglucose, 2 g of MgSO₄.7H₂O, 10 g of KH₂PO₄, 2 g of K₂SO₄, 2.08 g ofCaCl₂.2H₂O, 2 g of citric acid, 10 g of yeast extract, 0.5 g of AMGtrace metals, and 2 g of urea, final pH 6.0.

AMG trace metals is comprised per liter of 14.3 g of ZnSO₄.7H₂O, 2.5 gof CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g of FeSO₄.7H₂O, 8.5 g ofMnSO₄—H₂O, and 3 g of citric acid.

Aspergillus oryzae Fermentation Medium was composed per liter of 2 gMgSO₄.7H₂O, 2 g of KH₂PO₄, 3 g of K₂SO₄, 9 g of (NH4)₂HPO₄, 1 g ofcitric acid.H₂O, 10 g of yeast extract, 0.5 ml of AMG trace metals, 25 gof sucrose, and 0.55 mL of pluronic.

Aspergillus oryzae Feed Medium was composed per liter of 1 g of citricacid.H₂O, 320 g SatinSweet 65, and 5 g of pluronic.

MDU2BP medium is composed per liter of 45 g of maltose, 1 g ofMgSO₄.7H₂O, 1 g of NaCl, 2 g of K₂SO₄, 12 g of KH₂PO₄, 7 g of yeastextract, 2 g of urea, 0.5 ml of AMG trace metals solution.

Example 1 Fermentation and Mycelial Tissue

Trichoderma reesei RutC30 was grown under cellulase inducing standardconditions as described in the art (Mandels and Weber, 1969, Adv. Chem.Ser. 95: 391-413). Mycelial samples were harvested by filtration throughWhatman paper and quick-frozen in liquid nitrogen. The samples werestored at −80° C. until they were disrupted for RNA extraction.

Example 2 Expressed Sequence Tags (EST) cDNA Library Construction

Total cellular RNA was extracted from the mycelial samples described inExample 1 according to the method of Timberlake and Barnard (1981, Cell26: 29-37), and the RNA samples were analyzed by Northern hybridizationafter blotting from 1% formaldehyde-agarose gels (Davis et al., 1986,Basic Methods in Molecular Biology, Elsevier Science Publishing Co.,Inc., New York). Polyadenylated mRNA fractions were isolated from totalRNA with an mRNA Separator Kit™ (Clontech Laboratories, Inc., Palo Alto,Calif.) according to the manufacturer's instructions. Double-strandedcDNA was synthesized using approximately 5 μg of poly(A)+mRNA accordingto the method of Gubler and Hoffman (1983, Gene 25: 263-269), except aNotI-(dT)18 primer (Pharmacia Biotech, Inc., Piscataway, N.J.) was usedto initiate first strand synthesis. The cDNA was treated with mung beannuclease (Boehringer Mannheim Corporation, Indianapolis, Ind.) and theends were made blunt with T4 DNA polymerase (New England Biolabs,Beverly, Mass.).

BamH I/EcoR I adaptors were ligated to the blunt ends of the cDNA. Afterdigestion with NotI, the cDNA was size selected (ca. 0.7-4.5 kb) by 0.7%agarose gel electrophoresis using TAE buffer (4.84 g of Tris Base, 1.14ml of glacial acetic acid, and 2 ml of 0.5 M EDTA pH 8.0 per liter), andligated with pYES2 (Invitrogen Corporation, Carlsbad, Calif.) which hadbeen cleaved with Not I plus BamH/and dephosphorylated withcalf-intestine alkaline phosphatase (Boehringer Mannheim Corporation,Indianapolis, Ind.). The ligation mixture was used to transformcompetent E. coli TOP10 cells (Invitrogen Corporation, Carlsbad,Calif.). Transformants were selected on 2YT agar plates (Miller, 1992, AShort Course in Bacterial Genetics. A Laboratory Manual and Handbook forEscherichia coli and Related Bacteria, Cold Spring Harbor Press, ColdSpring Harbor, N.Y.) supplemented with ampicillin at a finalconcentration of 50 μg per ml.

Example 3 Template Preparation and Nucleotide Sequencing of cDNA Clones

From the cDNA library described in Example 2, approximately 7000transformant colonies were picked directly from the transformationplates into 96-well microtiter dishes which contained 100 μl of 2YTbroth supplemented with 50 μg of ampicillin per ml. The plates wereincubated overnight at 37° C. with shaking at 200 rpm. After incubation,100 μl of sterile 50% glycerol was added to each well. The transformantswere replicated into secondary, deep-dish 96-well microculture plates(Advanced Genetic Technologies Corporation, Gaithersburg, Md.)containing 1 ml of Magnificent Broth™ (MacConnell Research, San Diego,Calif.) supplemented with 50 μg of ampicillin per ml in each well. Theprimary microtiter plates were stored frozen at −80° C. The secondarydeep-dish plates were incubated at 37° C. overnight with vigorousagitation (300 rpm) on a rotary shaker. To prevent spilling andcross-contamination, and to allow sufficient aeration, each secondaryculture plate was covered with a polypropylene pad (Advanced GeneticTechnologies Corporation, Gaithersburg, Md.) and a plastic microtiterdish cover.

DNA was isolated from each well using a 96-well Miniprep Kit protocol ofAdvanced Genetic Technologies Corporation (Gaithersburg, Md.) asmodified by Utterback et al. (1995, Genome Sci. Technol. 1: 1-8).Single-pass DNA sequencing (EST) was performed with a Perkin-ElmerApplied Biosystems Model 377 XL Automated DNA Sequencer(Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif.) usingdye-terminator chemistry (Giesecke et al., 1992, Journal of VirologyMethods 38: 47-60) and a T7 sequencing primer:

(SEQ ID NO: 3) 5′-TAATACGACTCACTATAGGG-3′

Example 4 Analysis of DNA Sequence Data of cDNA Clones

Nucleotide sequence data were scrutinized for quality and vectorsequences and ambiguous base calls at the ends of the DNA sequences weretrimmed, and all sequences were compared to each other with assistanceof PHRED/PHRAP software (University of Washington, Seattle, Wash.). Theresulting contigs and singletons were translated in six frames andsearched against publicly available protein databases using GeneMatcher™software (Paracel, Inc., Pasadena, Calif.) with a modifiedSmith-Waterman algorithm using the BLOSUM 62 matrix.

Example 5 Identification of cDNA Clones Encoding a Family 7Cellobiohydrolase I (CeI7A)

Putative cDNA clones encoding a Family 7 cellobiohydrolase (CeI7A) wereidentified by comparing the deduced amino acid sequence of the assembledESTs to protein sequences deposited in publicly available databases suchas Swissprot, Genpept, and PIR. One clone, Trichoderma reesei ESTTr0221, was selected for nucleotide sequence analysis which revealed an1821 by pYES2 insert which contained a 1452 by open reading-frame asshown in SEQ ID NO: 1 and a deduced amino acid sequence as shown in SEQID NO: 2. The plasmid containing Trichoderma reesei CeI7Acellobiohydrolase I was designated pTr0221.

Example 6 Construction of Saccharomyces cerevisiae Vectors for theGeneration of Primary and Shuffled Trichoderma reesei CeI7ACellobiohydrolase I Libraries

Two vectors were utilized in the generation of primary and shuffledlibraries, pJC106 (WO9510602) and pAJ052 (FIG. 1). Plasmid pJC106 is aderivative of pYES2 (Invitrogen Inc., Carlsbad, Calif.) but differs inthat pJC106 has a full-length 2 micron replicon, which replaces thepartial 2 micron in pYES2, and contains the Coprinus cinereus peroxidase(CIP) (Cherry et al., 1999, Nat. Biotechnol. 4: 379-384) codingsequence, which is regulated by the GAL1 promoter.

For pAJ052, a 1452 by DNA fragment spanning from the ATG start codon tothe TAA stop codon of the Trichoderma reesei CeI 7A coding sequence wasPCR amplified from pTr0221 (Example 5) using primers aGal 776.1 (sense)and aGal_(—)776.1A (antisense) shown below:

Primer aGal_776.1: (SEQ ID NO: 4)5′-TATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAGGATCCACCATGTATCGGAAGTTGGCCG-3′ Primer aGal_776.1A: (SEQ ID NO: 5)5′-CATAACTAATTACATGATGCGGCCCTCTAGATGCACATGACTCGAGTTACAGGCACTGAG AGTAG-3′

Primers aGal_(—)776.1 and aGal_(—)776.1 A were designed to contain ahomologous GAL1 promoter (Giniger and Ptashne, 1988, Proc. Natl. Acad.Sci., USA 85: 382-386) and the CYC1 terminator sequence (Osbourne andGuarente, 1988, Genes Dev. 2: 766-772) (underlined, respectively) ofpJC106 for in vivo homologous recombination of the PCR product andpJC106. Primers aGal_(—)776.1 and aGal_(—)776.1 A were also designed tocontain BamH I and Xho I restriction sites, respectively. PrimeraGal_(—)776.1 was further designed to contain the yeast Kozak sequence(ACC, −3 to −1 bp; Kozak, 1984, Nature 308: 241-246) immediatelyupstream of the Trichoderma reesei cellobiohydrolase I (CeI7A) gene ATG.The amplification reaction (50 μl) was composed of 1×PCR buffer (AppliedBiosystems Inc., Foster City, Calif.), 0.2 mM dNTPs, 3.2 pM primeraGal_(—)776.1, 3.2 pM primer aGal_(—)776.1 A, approximately 100 ng ofpTr0221, and 2.5 units of Taq DNA Polymerase (Roche Applied Science,Manheim, Germany). The reactions were incubated in an EppendorfMastercycler 5333 (Eppendorf EG, Hamburg, Germany) programmed for 1cycle at 94° C. for 3 minutes followed by 30 cycles each at 94° C. for30 seconds, 55° C. for 30 seconds, and 72° C. for 90 seconds followed by1 cycle at 72° C. for 5 minutes. The PCR product was then purified usinga QIAquick PCR Kit (QIAGEN Inc., Valencia, Calif.), according to themanufacturer's instructions, and was then introduced into Saccharomycescerevisiae by in vivo recombination. To accomplish the in vivorecombination, approximately 100 ng of pJC016, digested with BamH I andXho I, and approximately 500 ng of the purified PCR fragment, wereco-transformed into Saccharomyces cerevisiae YNG 344 following theYEASTMAKER yeast transformation protocol (Clontech Laboratories Inc.,Palo Alto, Calif.). The transformation was plated onto yeast selectionmedium for colony growth at 30° C. for 4 days.

A single colony was selected and plasmid DNA was isolated according tothe protocol described by Kaiser and Auer, 1993, Bio Techniques 14: 552,which was subsequently transformed into E. coli strain XL-10 (StratageneInc., La Jolla, Calif.) according to the manufacturer's instructions.Plasmid derived from the transformed E. coli strain was sequenced toverify the fidelity of the PCR and recombination event.

Example 7 Generation of Primary Libraries of Mutagenized CeI7ACellobiohydrolase I in Saccharomyces cerevisiae

In an effort to identify regions of the Trichoderma reesei CeI7Acellobiohydrolase I that are critical for protein thermostability andimproved high-temperature activity, the entire wild-type Trichodermareesei CeI7A cellobiohydrolase I gene was mutagenized using error-pronePCR with homologous sequences to the yeast expression vector pJC106(FIG. 2), which can undergo in vivo recombination between homologousdomains of distinct fragments, generating circular, replicating plasmidsfrom a combination of linearized vector and PCR products.

PCR products for gap repair were generated using one of the followingtemplate/primer combinations:

1) Primer aGal_(—)776.1 and primer aGal_(—)776.1a, shown below, wereused in the error-prone PCR amplification of the CeI7A cellobiohydrolaseI gene from pTR0221 to generate mutagenized sequences.

2) Primer yes2term and primer CiPperdwn, shown below, were used in theerror-prone PCR amplification of the CeI7A cellobiohydrolase I gene frompAJ052 to generate mutagenized sequences.

3) Primer cJC106.1a and primer CiPperdwn, shown below, were also used inthe error-prone PCR amplification of the CeI7A cellobiohydrolase I genefrom pAJ052 to generate mutagenized sequences.

The fragments were cloned into pJC106 for expression of the CeI7Acellobiohydrolase I variants in yeast.

Primer aGal_776.1: (SEQ ID NO: 6)5′-TATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAGGATCCACCATGTATCGGAAGTTGGCCG-3′ Primer aGal_776.1a: (SEQ ID NO: 7)5′-CATAACTAATTACATGATGCGGCCCTCTAGATGCACATGACTCGAGTTACAGGCACTGAG AGTAG-3′Primer yes2term: (SEQ ID NO: 8) 5′-GGCGTGAATGTAAGCGTGAC-3′Primer CiPpcrdwn: (SEQ ID NO: 9) 5′-CTGGGGTAATTAATCAGCGAAGCGATGA-3′Primer cJC106.1 a: (SEQ ID NO: 10) 5′-GCGTACACGCGTCTGTACA-3′

The error-prone PCR amplifications (50 μl) were composed of 1×PCR bufferwith MgCl₂, 0.2 mM dATP, 0.2 mM dGTP, 0.1 mM dCTP and 0.1 mM dTTP, 50pmol of sense and antisense primer, 0.05 mM to 0.6 mM MnCl₂, and 10-50ng of plasmid DNA (in some cases pTR0221, in other cases pAJ052). Thereactions were incubated using a MJ Research thermocycler (MJ Research,Inc. Boston, Mass.) programmed for one cycle at 95° C. for 3 minutesafter which 2.5 units of Amplitaq (Perkin Elmer, Foster City, Calif.)were added followed by 30 cycles each at 95° C. for 60 seconds, 55° C.for 60 seconds, and 72° C. for 90 seconds. The reactions were thenincubated at 72° C. for a 5 minute extension. An aliquot of each PCRproduct was run on a 0.7% agarose gel using TAE buffer, as previouslydescribed, generating expected bands of approximately 1680 to 2030 bp.PCR reactions were purified using a MiniElute PCR Purification Kit(QIAGEN, Valencia, Calif.) eluted into 50 μl of EB buffer (QIAGEN,Valencia, Calif.).

Plasmid pJC106 was gapped by digestion with BamH I and Xho I, and thengel purified using a Qiaquick Minielute column (QIAGEN, Inc., Valencia,Calif.). The digestion was verified by fractionating an aliquot of thedigestion on a 0.8% agarose gel using TAE buffer and staining withethidium bromide where expected fragments of 10771 by (gapped) and 1030by (from the Coprinus cinereus peroxidase gene) were obtained.

The PCR reactions were mixed at approximately a 3 to 1 ratio with thegapped pJC106 vector for cotransformation into Saccharomyces cerevisiaeYNG344 competent cells. The co-transformed fragments, amplified usingprimers aGal_(—)776.1 and aGal_(—)776.1a, contained at least 67 by of 5′and 66 by of 3′ homologous DNA, amplified using yes2term and CiPperdwncontained at least 293 by of 5′ and 41 by of 3′ homologous DNA, andamplified using cJC106.1a and CiPperdwn contained at least 293 by of 5′and 190 by of 3′ homologous DNA at the ends to facilitate gap repair ofthe expressed plasmid. Competent cells of Saccharomyces cerevisiae YNG344 were prepared prior to each transformation following the YEASTMAKERYeast Transformation Protocol with the following modifications: (1) Thevolume of yeast culture used to inoculate the overnight incubation(16-20 hours) was between 100-1,000 μl; (2) recovery of cells upontransformation was performed in YPD medium for 45 minutes at 30° C.; and(3) the transformation mixture was aliquoted for plating onto yeastselection medium plates and frozen at −80° C. in a controlled ratefreezer (Nalge Nunc International, Rochester, N.Y.).

Example 8 Construction of pAILo1 and pAILo2 Aspergillus oryzaeExpression Vectors

As a backbone vector for cloning several of the CeI7A cellobiohydrolasevariants, two Aspergillus oryzae expression vectors were constructed.Vector pAlLo1 was constructed by modifying pBANe6 (U.S. Pat. No.6,461,837), which comprises the Aspergillus oryzae alpha-amylasepromoter (TAKA promoter), Aspergillus niger amyloglucosidase terminatorsequence (AMG terminator), and Aspergillus nidulans acetamidase gene(amdS). Modification of pBANe6 was performed by first eliminating threeNco I restriction sites at positions 2051, 2722, and 3397 by from theamdS selection marker by site-directed mutagenesis. All changes weredesigned to be “silent” leaving the actual protein sequence of the amdsgene product unchanged. Removal of these three sites was performedsimultaneously with a GeneEditor Site-Directed Mutagenesis Kit (Promega,Madison, Wis.) according to the manufacturer's instructions using thefollowing primers (underlined nucleotide represents the changed base):

Primer AMDS3NcoMut (2050): (SEQ ID NO: 11) 5′-GTGCCCCATGATACGCCTCCGG-3′Primer AMDS2NcoMut (2721): (SEQ ID NO: 12)5′-GAGTCGTATTTCCAAGGCTCCTGACC-3′ Primer AMDS1NcoMut (3396):(SEQ ID NO: 13) 5′-GGAGGCCATGAAGTGGACCAACGG-3′

A plasmid comprising all three expected sequence changes was thensubmitted to site-directed mutagenesis, using a QuickChange MutagenesisKit (Stratagene, La Jolla, Calif.), to eliminate the Nco I restrictionsite at the end of the AMG terminator at position 1643. The followingprimers (underlined nucleotide represents the changed base) were usedfor mutagenesis: Sense Primer to mutagenize the Aspergillus niger AMGterminator sequence:

(SEQ ID NO: 14) 5′-CACCGTGAAAGCCATGCTCTTTCCTTCGTGTAGAAGACCAGACAG-3′Antisense Primer to mutagenize the Aspergillus niger AMG terminatorsequence:

(SEQ ID NO: 15) 5′-CTGGTCTTCTACACGAAGGAAAGAGCATGGCTTTCACGGTGTCTG-3′

The last step in the modification of pBANe6 was the addition of a newNco I restriction site at the beginning of the polylinker using aQuickChange Mutagenesis Kit and the following primers (underlinednucleotides represent the changed bases) to yield pAlLo1 (FIG. 3). SensePrimer to mutagenize the Aspergillus oryzae TAKA promoter:

(SEQ ID NO: 16) 5′-CTATATACACAACTGGATTTACCATGGGCCCGCGGCCGCAGATC-3′Antisense Primer to mutagenize the A. oryzae TAKA promoter:

(SEQ ID NO: 17) 5′-GATCTGCGGCCGCGGGCCCATGGTAAATCCAGTTGTGTATATAG-3′

The amdS gene of pAlLo1 was swapped with the Aspergillus nidulans pyrGgene. Plasmid pBANe10 (FIG. 4) was used as a source for the pyrG gene asa selection marker. Analysis of the sequence of pBANe10 showed that thepyrG marker was contained within an Nsi I restriction fragment and doesnot contain either Nco I or Pac I restriction sites. Since the amdS wasalso flanked by Nsi I restriction sites, the strategy to switch theselection marker was a simple swap of Nsi I restriction fragments.

Plasmid DNA from pAlLo1 and pBANe10 were digested with the restrictionenzyme Nsi I and the products purified by 0.7% agarose gelelectrophoresis using TAE buffer. The Nsi I fragment from pBANe10containing the pyrG gene was ligated to the backbone of pAlLo1 toreplace the original Nsi I DNA fragment containing the amdS gene.Recombinant clones were analyzed by restriction enzyme digestion todetermine insert orientation. A clone with the pyrG gene transcribed inthe counterclockwise direction was selected. The new plasmid wasdesignated pAlLo2 (FIG. 5).

Example 9 Rational Design of Improved CeI7A Cellobiohydrolase I VariantG205R and Generation of G205R Primary Libraries in Saccharomycescerevisiae

The Trichoderma reesei CeI7A cellobiohydrolase I protein sequence (SEQID NO: 2) was compared to other proteins of the same enzyme family.Sequences included a CeI7A cellobiohydrolase I from Chaetomiumthermophilum (WO 03/000941), Humicola insolens (WO 95/02675), andNeurospora crassa (SWISSPROT: P38676a close phylogenetic relative ofChaetomium thermophilum). Multiple alignments of the CeI7Acellobiohydrolase I protein sequences from Chaetomium thermophilum,Humicola insolens, Trichoderma reesei, and Neurospora crassawere made,using ClustaIX software version 1.81, (National Center for BiotechnologyInformation, NIH Bethesda, Md.) (Thompson et al, 1994, Nucleic Acids Res22: 4673-4680; Thompson et al, 1997, Nucleic Acids Res 25: 4876-4882),using the Gonnet matrix with default gap penalty parameters. Regionsthat appeared poorly aligned were iteratively realigned, sometimes usingan alternative matrix (Blosum) and/or variable gap parameters. Homologymodels for publicly available CeI7A cellobiohydrolase I sequences weregenerated using the automated SwissModel service SwissModel service(Biozentrum, Basel, Switzerland). The homology model for the CeI7Acellobiohydrolase I from Humicola insolens was generated by using theInsight II programs (Accelrys, San Diego Calif.). The program DeepView(Guex and Peitsch, 1997, Electrophoresis 18: 2714-2723) was used tointroduce virtual mutations and for all other structure manipulationsand energy minimization. The reference structure used for the CeI7Acellobiohydrolase I from Trichoderma reesei was PDB: 7CEL (Divne et al,1994, Science 265: 524-528; Stahlberg et al, 1996, 264:337-349). Basedon the comparisons, potential mutations for Trichoderma reeseicellobiohydrolase I were prioritized based upon the likelihood that theywould create a new stabilizing interaction (ion pair and/or H-bond) andalso the probability of occurrence in the sequences and structures ofCeI7A cellobiohydrolase I of the thermophilic fungi Chaetomiumthermophilum and Humicola insolens, and their absence in the mesophilicfungus Neurospora crassa.

One of the amino acid substitutions suggested was a change from glycineat position 205 to arginine. The G205R variant was rationally designedto introduce ion-pairing with E190 and E239. To generate the G205Rsubstitution, the Trichoderma reesei CeI7A cellobiohydrolase I gene wassubcloned into the Aspergillus oryzae vector pAlLo02 digested with Nco Iand Pac Ito form a perfect junction with the ATG of the gene and theAspergillus oryzae alpha-amylase promoter and the Aspergillus nigeramyloglucosidase terminator sequence. Subcloning of the CeI7Acellobiohydrolase I gene into pAlLo2 was accomplished by designing twoprimers, shown below, that allowed cloning into the Nco I and Pac Isites. Primer cTR0221.7: 5′-GCAACATGTATCGGAAGTTGGC-3′ (SEQ ID NO: 18)incorporated a BspLU II site, which was compatible to the Nco I site inpAlLo2, to the 5′-end of the CeI7A cellobiohydrolase I gene, and primercTR0221.7a: 5′-AATTAATTTTACAGGCACTGAG-3′ (SEQ ID NO: 19) incorporated aBspLU II site at the-3′end.

Amplification of the CeI7A cellobiohydrolase I gene was accomplishedusing 1×Tgo Polymerase Reaction buffer (Boehringer Mannheim Co,Indianapolis, Ind.), 25 ng of pTR0221, 0.2 mM each of dATP, dGTP, dCTP,and dTTP, 50 pmole of each primer (cTR0221.7 and cTR0221.7a), and 1 unitof Tgo polymerase (Boehringer Mannheim Co, Indianapolis, Ind.). Thereactions were incubated using a MJ Research Thermocycler programmed forone cycle at 95° C. for 5 minutes, followed by 35 cycles each at 94° C.for 60 seconds, 55° C. for 45 seconds, and 72° C. for 2 minutes. Thereactions were then incubated at 72° C. for a 5 minute extension. Analiquot of each PCR product was run on a 0.7% agarose gel using TAEbuffer generating expected bands of approximately 1545 bp. The 1545 byPCR product was subcloned using a TOPO Blunt PCR4 Cloning Kit(Invitrogen, Carlsbad, Calif.). The resulting plasmid was digested withBspLU II and Pac I and fractionated on a 0.7% agarose gel using TAEbuffer generating an expected 1.5 kb coding sequence, which was excisedand gel purified using an Amicon Ultra-free DA column (Millipore,Billerica, Mass.). The resulting fragment was subsequently ligated intopAlLo2, which was digested similarly, to generate the expression vectordesignated pCW026 (FIG. 6) containing the Trichoderma reesei CeI7Acellobiohydrolase I gene.

Working from the starting plasmid pCW026, the G205R variant was obtainedby mutating a guanosine to cytidine at base 664 of the coding sequenceof CeI7A cellobiohydrolase I, using a Quick Change Site DirectedMutagenesis Kit (Stratagene, La Jolla, Calif.) with pCW026 as thetemplate. Primers used to incorporate this mutation were primer G205R.1and primer G205R.1a shown below:

G205R.1: (SEQ ID NO: 20) 5′-GAACACGGGCATTGGACGACACGGAAGCTGCTG-3′G205R.1a: (SEQ ID NO: 21) 5′-CAGCAGCTTCCGTGTCGTCCAATGCCCGTGTTC-3′The resulting expression vector was designated pNP776G205R (FIG. 7).

Example 10 Screening of CeI7A Cellobiohydrolase I Libraries

Primary CeI7A cellobiohydrolase I libraries were spread on agar yeastselection medium in Genetix QTrays (22×22 cm Petri dishes, GeneticsLtd., Hampshire, United Kingdom) and incubated for 5 days at 30° C.Using a Genetix QPix (Genetix Ltd., Hampshire, United Kingdom), colonieswere picked into 96-well plates containing yeast selection medium.Plates were incubated for 5-8 days at 30° C. Using an ORCA robot(Beckman Coulter, Fullerton, Calif.), the growth plates were transportedto a Biomek Fx (Beckman Coulter, Fullerton, Calif.) and broth sampleswere removed from the growth plate and aliquoted into two 96-wellpolycarbonate v-bottom plates. The cellobiohydrolase I substrate4-methylumbelliferyl-beta-D-lactoside (MUL, Marker Gene Tech. Inc.,Eugene, Oreg.) was added to each V-bottom 96-well plate to a finalconcentration of 0.2, 0.1, or 0.05 mg of4-methylumbelliferyl-beta-D-lactoside per ml, 0.1 M succinate pH 5.0,and 0.01% Tween-20. Assay plates were transferred to atemperature-controlled incubator, where one plate was incubated at 50°C. for 45 minutes, and another was incubated at a pre-determinedtemperature, between 62° C. and 65° C. for 45 minutes. After thisincubation, the plates were cooled to 4° C. for 1 minute, and thentransferred to the Biomek Fx where assays were quenched by addition ofTris-CI, pH 9.5 to a final concentration of 0.75 M. Quenched reactionsamples were diluted in water, and fluorescence of 4-methyl-umbelliferylliberated by CeI7A cellobiohydrolase I hydrolysis of4-methylumbelliferyl-beta-D-lactoside was measured using a BMG FLUOStarGalaxy fluorometer (Offenburg, Germany) (excitation 360 nm, emission 460nm). The ratio of the fluorescence from the plate treated at hightemperature (“high temperature activity”) was compared to fluorescencefrom the same samples incubated at 50° C. (“low temperature activity”),using Microsoft Excel (Microsoft Corporation, Redmond, Wash.) todetermine the relative thermal activity ratio for each variant. Based onthe thermal activity ratios, screening of libraries constructed inExample 7 and Example 9 generated the variants listed in Table 1. Table1 shows the degree of improvement for novel CeI7A cellobiohydrolase Ivariants as measured by assessing the thermal activity ratio of activityat 64° C. relative to activity at 50° C. For mutants obtained in theprimary screen, improvements range from 2.60-fold higher to 10.20-foldimprovement relative to the wild type enzyme. For mutants obtained fromshuffling, the improvement observed was 12.40-fold to 19.20-fold betterthermal activity than the wild type enzyme.

TABLE 1 Cel7A variants with improved thermal stability and thermalactivity. “Fold Improvement” indicates relative improvement in thermalactivity ratio, measured at 64° C./50° C. Cel7A IFold Variant Amino AcidSubstitutions* Improvement Wild type none 1 G205R G205R 2.60 776-M1T226A 6.20 776-M3 P227A, C486W 7.80 776-M4 S113N, S196T, T462A 6.00776-M21 N301S, E337V 4.00 776-M22 S196P, T350S 1.60 776-M23 G22D, G467S1.40 776-M26 S21P, S57N 3.80 776-M27 S411F 7.60 776-M30 T41I 2.60776-M32 K157R, G205R, T255P 5.20 776-M35 G205R, S411F 8.40 776-M40G205R, P227A 10.20 776-M41 G205R, H206Y 3.40 776-M42 S8P, G205R 4.60776-M52 G94S, G205R 4.00 776-M53 S196P, G205R 4.80 776-M57 S113N, S196T,P227A, T462A 14.60 776-M65 S57N 5.00 776-M71 T383A, T455A 2.60 776-M73N373H 4.80 776-M101 S113N, S411F 12.40 776-M108 T41I, E193K, S411F 15.60776-M109 N49S, S113N, P227A, P438L 15.00 776-M124 Y247C, Y371C, S411F17.40 776-M125 S21P, S57N, T246I, R251K, S411F 18.00 776-M192 K157R,G205R, T255P, S411F 15.00 776-M216 S113N, S196T, P227A, S411F 17.80776-M252 S113N, S196T, P227A, T356I, T462A 19.20 *Numbering uses thefirst residue of the mature Cel7A enzyme as position 1.

Example 11 DNA Sequencing of Variants

To determine the sequence of the CeI7A cellobiohydrolase I variantsderived from the libraries of Examples 7 and 9, plasmid DNA wasisolated. Each variant was streaked onto agar yeast selection medium andincubated for 3-5 days at 30° C. Eight colonies were isolated andinoculated into 1 ml of yeast screening medium, and grown for 5-8 daysat 30° C. The agar plates were re-grown at 30° C. for 3-5 days. Culturebroth from the single colonies of each variant was assayed for improvedthermal activity of the produced cellobiohydrolase I variant asdescribed in Example 10 to determine which had improved thermal activityratios relative to wild type (wt) CeI7A cellobiohydrolase I. Plasmid wasrescued from the colonies which produced cellobiohydrolase I variantswith improved thermal activity as described by Kaiser and Auer, 1993,Bio Techniques 14: 552, using E. coli strain XL-10 (Stratagene Inc., LaJolla, Calif.).

DNA sequencing was performed using an ABI 3700 sequencer (AppliedBiosystems, Foster City, Calif.) using dye terminator chemistry(Giesecke et al., 1992, Journal of ViroL Methods 38: 47-60). Plasmid DNAfor sequencing was prepared using a BioRobot 9604 (QIAGEN, Valencia,Calif.). The entire coding region for each Trichoderma reesei CeI7Acellobiohydrolase I variant was sequenced using 0.5 μl of plasmid DNAand 3.2 pmol of the following primers:

cTr0221.1: (SEQ ID NO: 22) 5′-CTTCTTGGCCACAGCTCGTG-3′ cTr0221.2:(SEQ ID NO: 23) 5′-GGCTTTGTCACCCAGTCTGC-3′ cTr0221.3: (SEQ ID NO: 24)5′-CGTCATCCAACAACGCGAAC-3′ cTr0221.4: (SEQ ID NO: 25)5′-TTCGAGACGTCGGGTGCCAT-3′ cTr0221.4: (SEQ ID NO: 26)5′-CGCGGAAGCTGCTCCACCAG-3′ cTr0221.1A: (SEQ ID NO: 27)5′-AATGGAGAGGCTGTTACCGC-3′Sequence trace files were edited and assembled using Vector NTI ContigExpress (Informax, Inc., Bethesda, Md.).

Example 12 Generation of Error Prone Libraries Using Variant CeI7ACellobiohydrolase I Genes as Templates

Random mutagenesis of several of the improved variants was performed.Plasmid DNA was used that had been rescued from yeast as described inExample 10, and error-prone PCR was performed as described in Example 7.In addition, the G205R variant was mutagenized by using pNP776G205R as atemplate in error-prone PCR. The following primers were used to generatemutagenized sequences:

Primer aGal_776.1: (SEQ ID NO: 28) 5′ TATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAGGATCCACCATGTATCGGAAGTTGGCCG-3′ Primer aGal_776.1a: (SEQ ID NO: 29)5′ CATAACTAATTACATGATGCGGCCCTCTAGATG CACATGACTCGAGTTACAGGCACTGAGAGTAG-3′

The error-prone PCR amplifications were analyzed on 0.7% agarose gelsusing TAE buffer and transformed with gapped pJC106 vector intoSaccharomyces cerevisiae as described in Example 7.

Variants 76-M35, 776-M40, 776-M41, 776-M42, 776-M52, and 776-M53 wereobtained from screening libraries created by error-prone amplificationof the G205R template. Variant 776-252 was derived from a librarycreated by mutagenic amplification of variant 776-M57. DNA from thesevariants was rescued and sequenced as described in Example 10.

Example 13 Shuffled libraries of CeI7A cellobiohydrolase I

To shuffle the CeI7A cellobiohydrolase I variants derived frommutagenesis of wild type CeI7A cellobiohydrolase I and G205R templates,plasmid DNA was isolated from the variants as described in Example 10.The CeI7A cellobiohydrolase I genes were amplified from the variantsusing the following primers:

Primer CiPpcrdwn: (SEQ ID NO: 30) 5′-CTGGGGTAATTAATCAGCGAAGCGATGA-3′Primer cJC106.1A: (SEQ ID NO: 31) 5′-GCGTACACGCGTCTGTACA-3′

Each amplification reaction (50 μl) was composed of 1×PCR buffer, 0.2 mMdNTPs, 3.2 pM primer aGal_(—)776.1, 3.2 pM primer aGal_(—)776.1 A,approximately 100 ng of pTr0221, and 2.5 units of Taq DNA polymerase.The reactions were incubated in an Eppendorf Mastercycler 5333programmed for 1 cycle at 94° C. for 3 minutes followed by 30 cycleseach at 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 90seconds (5 minute final extension). PCR products were then purifiedusing a QIAquick PCR Kit according to the manufacturer's instructions.

For shuffling of the CeI7A cellobiohydrolase I variants, PCR productsfrom the amplified variant genes were combined with approximately 50 ngof pJC016 digested with BamH I and Xho I, and 100-200 ng of the assortedPCR fragments were co-transformed into Saccharomyces cerevisiae YNG 344following the YEASTMAKER yeast transformation protocol to generateshuffled libraries, as described in Example 7. A large number ofshuffled libraries were generated. The total number of variants includedin a single library ranged from two to ten. The percentage of activevariants in these libraries ranged from 87% to 94%, based on 50° C.activity determined in the 4-methylumbelliferyl-beta-D-lactoside assaydescribed in Example 9. Since the Taq DNA polymerase used has lowproofreading activity, the process of shuffling may have introduced newmutations in the CeI7A cellobiohydrolase I coding sequence.

Colonies from the shuffled libraries were picked and screened asdescribed in Example 9, using a comparison of the 64° C. activity to the50° C. activity using 4-methylumbelliferyl-beta-D-lactoside as substrateto assess the degree of improved thermal stability and thermal activityof each variant.

Screening of the shuffled libraries resulted in the isolation ofimproved variants, designated 776-M57, 776-M101, 776-M108, 776-M109,776-M124, 776-M125, 776-M192, and 776-M216. DNA from these variants wasrescued and sequenced as described in Example 10. Some of thesubstitutions identified by DNA sequencing of variants 776-M57,776-M101, 776-M108, 776-M109, 776-M124, 776-M125, 776-M192, and 776-M216contain substitutions previously identified by sequencing the mutantsfrom error-prone libraries. For example, 776-M57 contained the S113Nmutation that was also found in one parent (776-M4) of the shuffledlibrary from which it was derived. Likewise, 776-M101 contained S411F, asubstitution found in one of the parents (776-M27) of the shuffledlibrary from which it was obtained. Other substitutions found in thevariants derived from shuffled libraries, including N49S, E193K, R251K,T246I, Y247C, Y371C, and P438L, were not observed in the sequencedvariants that comprised the parent variants for the correspondingshuffled libraries. These mutations may have been introduced by aPCR-mediated mutagenesis event during amplification of the variant DNAduring construction of the shuffled libraries.

Example 14 Site-Specific Saturation Mutagenesis

To screen for the optimal substitution for improved thermal activity atspecific positions in CeI7A cellobiohydrolase I, site-specificrandomization was performed. Amino acids G94, S196, and P227 wererandomized by substitution of the wild-type codon with NN (G/C) usingmegaprimer PCR (Landt, et al., 1990 Gene 96: 125-128). Randomization atthe three positions was performed using the following primers:

Randomization at position 94:

Primer aTrCBHI.2: (SEQ ID NO: 32) 5′-GCGGTAACAGCCTCTCCATTNNSTTTGTCAC-3′Primer aTrCBHI.2a: (SEQ ID NO: 33)5′-CTGCGCAGACTGGGTGACAAASNNAATGGAGAG-3′Randomization at position 196:

Primer aTrCBHI.3: (SEQ ID NO: 34) 5′-CCATCTCCGAGGCTCTTACCNNSCACCCTTGC-3′Primer aTrCBHI.3a: (SEQ ID NO: 35)5′-GGCCGACAGTCGTGCAAGGGTGSNNGGTAAGAG-3′Randomization at position 227:

Primer aTrCBHIR.1: (SEQ ID NO: 36) 5′-GAGGGCTGGGAGCCGTCANNSAACAACGCG-3′Primer aTrCBHI.1bA: (SEQ ID NO: 37)5′-CCAATGCCCGTGTTCGCGTTGTTSNNTGACGGC-3′

Each amplification reaction (50 μl) was composed of 1×PCR buffer, 0.2 mMdNTPs, 3.2 pM of the sense primer, 3.2 pM of the antisense primer,approximately 100 ng of pTr0221, and 2.5 units of Taq DNA Polymerase.The reactions were incubated in an Eppendorf Mastercycler 5333programmed for 1 cycle at 95° C. for 3 minutes followed by 30 cycleseach at 95° C. for 30 seconds, 55° C. for 60 seconds, and 72° C. for 90seconds (5 minute final extension). PCR products were then purifiedusing a QIAquick PCR Kit according to the manufacturer's instructions.Each PCR fragment generated for the individual site mutations served asmegaprimers for the overlap extension amplification reaction to generatea complete cellobiohydrolase I gene fragment, with indicatedsite-specific mutations, for library generation. The first 5 rounds ofthe amplification reaction was composed of 1×PCR buffer, 0.2 mM dNTPs,100 ng of the sense (5′) megaprimer, 100 ng of the antisense (3′)megaprimer, and 2.5 units of Taq DNA Polymerase. The reactions wereincubated in an Eppendorf Mastercycler 5333 programmed for 1 cycle at95° C. for 3 minutes followed by 5 cycles each at 95° C. for 30 seconds,55° C. for 60 seconds, and 72° C. for 90 seconds. To this reaction, 50pmol of primer aGal776.1 and aGal776.1A were added to amplify the newlygenerated site randomized fragment generated in the previous 5 cyclereaction. The reactions after the addition of the new primers wereincubated in an Eppendorf Mastercycler 5333 programmed for 30 cycleseach at 95° C. for 30 seconds, 55° C. for 60 seconds, and 72° C. for 90seconds (5 minute final extension). The resulting PCR fragments werethen purified using a QIAquick PCR Kit according to the manufacturer'sinstructions. The final PCR fragment was then transformed directly intoyeast together with BamH I and Xho I-gapped pJC106 plasmid (Example 6).

Since the Taq DNA polymerase used has low proofreading activity, theprocess of saturation mutagenesis may have introduced new mutations inthe coding sequence in addition to the designed changes at positions 94,196, and 227. Libraries containing site-specific randomized amino acidswere screened as described in Example 10. As shown in Table 2, a numberof substitutions that improved the thermal activity ratio at 63° C./50°C. relative to wild type were discovered in addition to thesubstitutions identified by screening randomly-mutagenized libraries.For example, variant 776-M3 was obtained from screening a primarylibrary, where a proline at position 227 was converted to an alanine. Inscreening the site-specific randomized library at this position, weindentified substitutions to leucine and to glycine as well as alanine.

In addition to uncovering the amino acid substitutions at positions 227,94, and 196, which confer improved thermal stability and activity, asecond site mutation was identified that could improve thermal activity.Specifically, the substitution D259N was identified in variant 776-M273as shown in Table 2.

TABLE 2 Cel7A variants with improved thermal stability containingsubstitutions at positions 227, 94, and 196. “Fold Improvement”indicates relative improvement in thermal activity ratio, measured at63° C./50° C. Cel7A Amino Acid Fold- Variant Substitutions* ImprovementLibrary Type Wild type none 1 none 776-M3 P227A, C486W 2.67 primary776-M259 P227L 1.50 Site-specific randomization at 227 776-M273 P227G,D259N 3.00 Site-specific randomization at 227 776-M274 P227A 2.67Site-specific randomization at 227 776-M275 P227L 2.00 Site-specificrandomization at 227 776-M52 G94S, G205R 2.00 primary 776-M268 G94A,T226A 2.67 Site-specific randomization at 94 776-M264 G94R 2.07Site-specific randomization at 94 776-M266 G94Q 2.17 Site-specificrandomization at 94 776-M269 G94A 2.03 Site-specific randomization at 94776-M53 S196P, G205R 1.50 primary 776-M4 S113N, S196T, n.a. primaryT462A 776-M261 S196P 1.33 Site-specific randomization at 196 776-M263T41I, S196F 1.67 Site-specific randomization at 196 *Numbering uses thefirst residue of the mature Cel7A enzyme as position 1.

Example 15 Construction of pMJ04, pMJ06, and pMJ09 Expression Vectors

Expression vector pMJ04 was constructed by PCR amplifying theTrichoderma reesei cellobiohydrolase 1 gene (cbh1) terminator fromTrichoderma reesei RutC30 genomic DNA using primers 993429 (antisense)and 993428 (sense) shown below. The antisense primer was engineered tohave a Pac I site at the 5′-end and a Spe I site at the 3′-end of thesense primer.

Primer 993429 (antisense): (SEQ ID NO: 38)5′-AACGTTAATTAAGGAATCGTTTTGTGTTT-3′ Primer 993428 (sense):(SEQ ID NO: 39) 5′-AGTACTAGTAGCTCCGTGGCGAAAGCCTG-3′

Trichoderma reesei RutC30 genomic DNA was isolated using a DNeasy PlantMaxi Kit (QIAGEN, Valencia, Calif.). The amplification reactions (50 μl)were composed of 1×ThermoPol Reaction Buffer (New England Biolabs,Beverly, Mass.), 0.3 mM dNTPs, 100 ng of Trichoderma reesei RutC30genomic DNA, 0.3 μM primer 993429, 0.3 μM primer 993428, and 2 units ofVent polymerase (New England Biolabs, Beverly, Mass.). The reactionswere incubated in an Eppendorf Mastercycler 5333 programmed as follows:5 cycles each for 30 seconds at 94° C., 30 seconds at 50° C., and 60seconds at 72° C., followed by 25 cycles each for 30 seconds at 94° C.,30 seconds at 65° C., and 120 seconds at 72° C. (5 minute finalextension). The reaction products were isolated on a 1.0% agarose gelusing TAE buffer where a 229 by product band was excised from the geland purified using a QIAquick Gel Extraction Kit (QIAGEN, Valencia,Calif.) according to the manufacturer's instructions.

The resulting PCR fragment was digested with Pac I and Spe I and ligatedinto pAlLo01 (Example 8) digested with the same restriction enzymesusing a Rapid Ligation Kit (Roche, Indianapolis, Ind.) to generate pMJ04(FIG. 8).

Expression vector pMJ06 was constructed by PCR amplifying theTrichoderma reesei CeI7A cellobiohydrolase 1 gene (cbh1) promoter fromTrichoderma reesei RutC30 genomic DNA using primers 993696 (antisense)and 993695 (sense) shown below. The antisense primer was engineered tohave a Sal I site at the 5′-end of the sense primer and an Nco I site atthe 5′-end of the antisense primer.

Primer 993695 (sense): (SEQ ID NO: 40) 5′-ACTAGTCGACCGAATGTAGGATTGTT-3′Primer 993696 (antisense): (SEQ ID NO: 41) 5′-TGACCATGGTGCGCAGTCC-3′

The amplification reactions (50 μl) were composed of 1×ThermoPolReaction Buffer, 0.3 mM dNTPs, 100 ng of Trichoderma reesei RutC30genomic DNA (which was prepared using a DNeasy Plant Maxi Kit, 0.3 μMprimer 993696, 0.3 μM primer 993695, and 2 units of Vent polymerase. Thereactions were incubated in an Eppendorf Mastercycler 5333 programmed asfollows: 30 cycles each for 30 seconds at 94° C., 30 seconds at 55° C.,and 60 seconds at 72° C. (15 minute final extension). The reactionproducts were isolated on a 1.0% agarose gel using TAE buffer where a988 by product band was excised from the gel and purified using aQlAquick Gel Extraction Kit according to the manufacturer'sinstructions.

The resulting PCR fragment was digested with Nco I and Sal I and ligatedinto pMJ04 digested with the same restriction enzymes using a RapidLigation Kit to generate pMJ06 (FIG. 9).

Expression vector pMJ09 was constructed by PCR amplifying theTrichoderma reesei CeI7A cellobiohydrolase 1 gene (cbh1) terminator fromTrichoderma reesei RutC30 genomic DNA using primers 993843 (antisense)and 99344 (sense) shown below. The antisense primer was engineered tohave a Pac I and a Spe I sites at the 5′-end and a Pvu I site at the5′-end of the sense primer.

Primer 993844 (sense): (SEQ ID NO: 42) 5′-CGATCGTCTCCCTATGGGTCATTACC-3′Primer 993843 (antisense): (SEQ ID NO: 43)5′-ACTAGTTAATTAAGCTCCGTGGCGAAAG-3′

The amplification reactions (50 μl) were composed of 1× ThermoPolReaction Buffer, 0.3 mM dNTPs, 100 ng Trichoderma reesei RutC30 genomicDNA (which was extracted using a DNeasy Plant Maxi Kit), 0.3 μM primer993844, 0.3 μM primer 993843, and 2 units of Vent polymerase. Thereactions were incubated in an Eppendorf Mastercycler 5333 programmed asfollows: 30 cycles each for 30 seconds at 94° C., 30 seconds at 55° C.,and 60 seconds at 72° C. (15 minute final extension). The reactionproducts were isolated on a 1.0% agarose gel using TAE buffer where a473 by product band was excised from the gel and purified using aQIAquick Gel Extraction Kit according to the manufacturer'sinstructions.

The resulting PCR fragment was digested with Pvu I and Spe I and ligatedinto pMJ06 digested with Pac I and Spe I using a Rapid Ligation Kit togenerate pMJ09 (FIG. 10).

Example 16 Construction of pCW045 for Expression of Variant 776-M57 inTrichoderma reesei

Variant 776-M57 was subcloned into the Trichoderma reesei expressionvector pMJ09 using an In-Fusion PCR Cloning Kit (BD Biosciences,Clonetech, Palo Alto, Calif.). PCR amplification of the variants wasperformed using Platinum Pfx DNA Polymerase (Invitrogen, Carlsbad,Calif.). Amplifications were composed of 1×Pfx amplification buffer, 0.3mM each dCTP, dATP, dTTP, and dGTP; 1 mM MgSO₄, 1 pmol of primer IF-F1,1 pmol of primer IF-R1, 50-100 ng of DNA template 776-57, and 1 unit ofPfx polymerase. The reactions were incubated using a MJ ResearchThermocycler programmed for one cycle at 95° C. for 3 minutes followedby 30 cycles each at 95° C. for 15 seconds, 55° C. for 30 seconds, and68° C. for 2 minutes 30 seconds. The reactions were then incubated at72° C. for a 7 minute extension.

Primer IF-F1: (SEQ ID NO: 44) 5′-CGCGGACTGCGCACCATGTATCGGAAGTTG-3′Primer IF-R1: (SEQ ID NO: 45) 5′-CGCCACGGAGCTTAATTACAGGCACTGAGA-3′

An aliquot of each PCR product was run on a 0.7% agarose gel using TAEbuffer, as previously described, generating expected bands ofapproximately 1.575 kb. PCR reactions were purified using a MinElute PCRPurification Kit and eluting the DNA into 50 μl of EB buffer. The yieldof each purified PCR product was estimated to be 125 ng per microliterby visualization on a 0.7% agarose gel using TAE buffer.

Plasmid pMJ09 digested with Pac I and blunted at the Nco I site waspurified using a Qiaquick Minielute Column Kit (QIAGEN, Valencia,Calif.). The plasmid had a concentration of 100 ng per microliter. Theconcentration was verified by visualization on a 0.7% agarose gel usingTAE buffer. Cloning of the 776-M57 PCR product described above and thedigested pMJ09 vector was accomplished by using the In-Fusion PCRCloning Kit (BD Biosciences, Clonetech, Palo Alto, Calif.). Theresulting Trichoderma reesei expression vector containing variant776-M57 was designated pCW045 (FIG. 11).

Example 17 Expression CeI7A Cellobiohydrolase I in Trichoderma reesei asthe Sole Cellobiohydrolase I

In order to evaluate the 776-M57 variant as the sole cellobhiohydrolaseI following expression in the native host, a Trichoderma reesei strainwas constructed wherein the cellobiohydrolase I CeI7A gene, cbh1, hadbeen disrupted. A disruption cassette was constructed using thehygromycin resistance gene (hph) from Escherichia coli as a selectablemarker. The hph marker was flanked by homologous cbh1 sequence in orderto target the native cbh1 gene.

Amplification of the genomic cbh1 gene was done using the polymerasechain reaction method with PWO Polymerase (Roche Applied Science,Manheim, Germany). The reactions were incubated in an EppendorfMastercycler 5333. Amplifications were composed of 1×PWO amplificationbuffer, 0.3 mM each of dCTP, dATP, dTTP, and dGTP, 1 mM MgSO₄, 1 pmoleach of primer cbh1 N-term and cbh1 C-term described below, and 50-100ng of DNA template. Amplifications utilized an initial denaturation of 2minutes at 95° C. followed by 35 cycles of a 1 minute denaturation, 2minute annealing at 55° C. and a 2 minute extension at 68° C.

Primer cbh1 N-term: (SEQ ID NO: 46) 5′-GCC TTC GGC CTT TGG GTG TA-3′Primer cbh1 C-term: (SEQ ID NO: 47) 5′-GAG CGG CGA TTC TAC GGG TT-3′

The 2.3 kb PCR fragment was cloned using the Zero Blunt TOPO PCR CloningKit (Invitrogen Corporation, Carlsbad, Calif.) and propagated in E. coliTop10 cells (Invitrogen Corporation, Carlsbad, Calif.). The resultingconstruct was a 5.8 kb plasmid designated pSTMO1 (FIG. 12).

The hph gene was amplified from the PHT1 plasmid (Cummings, et al.,1999, Curr. Genet. 36: 371-382) using the polymerase chain reactionmethod with PWO Polymerase. The reactions were incubated in an EppendorfMastercycler 5333, and were composed of 1×PWO amplification buffer, 0.3mM each of dCTP, dATP, dTTP, and dGTP, 1 mM MgSO₄, 1 pmol each of primerhph N-term and hph C-term described below, and 50-100 ng of DNAtemplate. The amplifications utilized an initial denaturation of 2minutes at 95° C. followed by 35 cycles of a 1 minute denaturation, 2minute annealing at 55° C. and a 2 minute extension at 68° C.

Primer hph N-term: (SEQ ID NO: 48)5′-GCC GCG GCA CGC GCC ACA CGG AAA AT-3′ Primer hph C-term:(SEQ ID NO: 49) 5′-GAC CGG TCG CAA AAT GAC AAA TAG AAG-3′

To generate the cbh1 disruption cassette, pSTMO1 was digested with Mlu Iand BstE II to create a 1.1 kb gap in the cbhl coding sequence. T4 DNApolymerase (New England Biolabs, Beverly, Mass.) was then used to fillin and generate blunt ends in the digested plasmid. The T4 reaction wasincubated for 20 minutes at 15° C. followed by a heat inactivation for15 minutes at 65° C. The entire reaction mix was fractionated on a 1%agarose gel using TAE buffer, and the major band was excised and elutedfrom the gel using a Qiaquick Gel Extraction Kit. The 1.7 kb hph genewas then inserted into the gapped pSTMO1 vector using a Rapid DNALigation Kit and transformed into One Shot E. coli competent cells(Invitrogen Corporation, Carlsbad, Calif.). The resulting plasmid wasdesignated pSMKO3 (FIG. 13).

Using pSMKO3 as a template, cbhl DNA was amplified using PCR primersflanking the hph gene. A 2.7 kb fragment was amplified from pSMKO3 usingthe following primers:

Primer pSMK03-F1: (SEQ ID NO: 50) 5′-GCTCCGGGCAAATGCAAA GTG TG-3′Primer pSMK03-R1: (SEQ ID NO: 51) 5′-AGCAGGCCGCATCTCCAGTGAAAG-3′

The amplification was performed using the polymerase chain reactionmethod with PWO Polymerase. The reactions were incubated in an EppendorfMastercycler 5333, and were composed of 1×PWO amplification buffer, 0.3mM each of dCTP, dATP, dTTP, and dGTP, 1 mM MgSO₄, 1 pmol each of primerKO N-term and KO C-term described above, and 50-100 ng of DNA template.Amplifications utilized an initial denaturation of 2 minutes at 95° C.followed by 35 cycles of a 1 minute denaturation, 2 minute annealing at55° C., and a 2 minute extension at 68° C. The resulting DNA fragmentcontained 639 kb of the cbhl promoter/gene sequence, the 1.7 kb of thehph coding region, and 659 bases of the cbhl gene/terminator sequence.

Protoplasts of Trichoderma reesei RutC30 were prepared fortransformation by the following method. Shake flasks containing 25 ml ofYPD medium were inoculated with 5×10⁷ conidia. Following an overnight(approximately 18 hours) incubation at 34° C. (150 rpm), the myceliawere collected by filtration through sterile Miracloth™ (Calbiochem, SanDiego, Calif.) and transferred to 20 mg/ml Glucanex (Novozymes A/S,Bagsvrd, Denmark) and 1.6 mg/ml chitinase (Sigma-Aldrich, St. Louis,Mo.) in 25 ml of 1 M sorbitol. Digestions were typically 25-40 minuteswith gentle shaking (80-100 rpm) at 34° C. The protoplasts were thenfiltered through a gauze filter and washed with ice cold 1 M sorbitoland then centrifuged at 400×g for 8 minutes. Protoplasts were washedtwice with 25 ml of 1 M sorbitol and twice with 25 ml of 1 M sorbitol,50 mM Tris-HCl pH 8.0, 50 mM CaCl₂. Protoplasts were then counted usinga haemacytometer and resuspended in a solution composed of 7 parts of 1M sorbitol, 50 mM Tris-HCl pH 8.0, 50 mM CaCl₂ and 2 parts of 50%PEG-4000, 50 mM Tris-HCl pH 8.0, 50 mM CaCl₂ at a concentration of 1×10⁸protoplasts/ml. Protoplasts were either used immediately or stored at−80° C. in a controlled rate freezer (Nalge Nunc International,Rochester, N.Y.) until transformation.

Transformation of the protoplasts was performed using 5 μg of linear DNAand 5 μl of heparin (5 mg/ml), which were mixed and incubated on ice for5 minutes. One hundered microliters of protoplasts were then added andthe mixture was incubated for 15 minutes on ice. Five hundredmicroliters of 50% PEG-4000, 50 mM Tris-HCl pH 8.0, 50 mM CaCl₂ was thenadded and incubated at room temperature for 15 minutes. One milliliterof 50% PEG-4000, 50 mM Tris-HCl pH 8.0, 50 mM CaCl₂ was added to themixture and incubated at 34° C. for 20 minutes. Following the incubationat 34° C., 2 ml of 1 M sorbitol, 50 mM Tris-HCl pH 8.0, 50 mM CaCl₂ wasadded. The contents were gently mixed before spreading 350 μl of theprotoplast mixture on PDA plates. The plates were incubated at 29° C.for approximately 18 hours before overlaying with 100 μg/ml hygromycin B(Roche Applied Science, Manheim, Germany) in PDA medium. Transformantsappeared within 3 days of the hygromycin overlay. Transformants werethen subcultured to PDA plates containing 100 μg/ml hygromycin B.Transformants that grew on the secondary plates were then used forinoculation into cellulase inducing medium (CIM).

Strains were screened for cellobiohydrolase I disruption by thefollowing method. Transformants were grown in cellulase-inducing mediumin 24-well tissue culture plates (Corning, Acton, Mass.) for 3 days at34° C. Ten microliters of culture broth was added to 10 μl of 2× samplebuffer plus 1% beta-mercaptoethanol and run on an 8-16% Tris-glycineacrylamide gel (NUPAGE Novex Gels Invitrogen Corporation, Carlsbad,Calif.) using NuPAGE® MES SDS Running Buffer (NUPAGE Novex GelsInvitrogen Corporation, Carlsbad, Calif.). Potential knockouts wereselected based on the absence of a cellobiohydrolase I CeI7A proteinband in comparison to the parent strain.

To confirm deletion of the cbh1 gene in candidate strains that had beenselected by SDS-PAGE analysis, Southern analysis was performed to assesswhether the cbh1 gene had been disrupted. Mycelia were grown overnightin shake flasks containing 25 ml of YPD medium. The mycelia were thenharvested, filtered, and ground using a mortar and pestle with liquidnitrogen. Genomic DNA was isolated using a DNeasy Plant Maxi Kit. Onemicrogram of DNA was digested either with Hind III or Nde I and run on a0.8% agarose gel in TAE buffer. The DNA was fragmented in the gel bytreating with 0.25 M HCl, denatured with 0.5 M NaOH, 1.5 M NaCl, andneutralized with 1 M Tris, pH 8.0; 1.5 M NaCl for subsequent transfer in10×SSC to Nytran Plus membrane (Schleicher & Schuell BioScience, GmbH,Dassel, Germany). The DNA was UV-crosslinked to the membrane andprehybridized for 1 hour at 60° C. in 20 ml of DIG Easy Hyb (RocheApplied Science, Manheim, Germany).

Two probes were prepared for Southern analysis. A 1.7 kb hygromycinprobe was amplified from pSMK03, using polymerase chain reaction and thefollowing primers:

Primer cpht-p: (SEQ ID NO: 52) 5′-GCACGCGCCACACGGAAAAT-3′ Primer cpht-t:(SEQ ID NO: 53) 5′-CGCAAAATGACAAATAGAAG-3′

In addition, a 2.1 kb cellobiohydrolase I probe was amplified frompSTMO1 by PCR using the primers pSMK03-F1 and pSMK03-R1. Probes wereprepared with a PCR DIG Probe Synthesis Kit (Roche Applied Science,Manheim, Germany). The PCR DIG Probe Synthesis mix and PCR buffer withmagnesium were used at 1×. Expand High Fidelity Polymerase (RocheApplied Science, Manheim, Germany) was added at 0.75 μl per reaction.One pmol each of primers and 50-100 ng of DNA template were used perreaction. Prior to use the probe was denatured by boiling for 5 minutesand then added to the hybridization buffer.

The denatured probe was added directly to the DIG Easy Hyb buffer andhybridization was overnight at 65° C. Following the post hybridizationwashes (twice in 2×SSC, once in 0.4×SSC, 60° C., 10 minutes each),chemiluminescent detection using the DIG detection system and CPD-Star(Roche Applied Science, Manheim, Germany) was done. The DIG-labeled DNAMolecular Weight Marker III Kit (Roche Applied Science, Manheim,Germany) was used as the source of standard markers.

Southern analysis indicated that the presence of a single hphintergration within the cbh1gene for a specific transformant. Thiscellobiohydrolase I gene deletion strain was designated Trichodermareesei SaMe013.

Plasmid pCW045, containing the cellobiohydrolase I variant 776-M57behind the cbh1 promoter, was introduced into Trichoderma reesei SaMe013by PEG-mediated transformation (Penttila et al., 1987, Gene 61:155-164). The plasmid contains the Aspergillus nidulans amdS gene toenable transformants to grow on acetamide as the sole nitrogen source.Trichoderma reesei RutC30 was cultivated at 27° C. and 90 rpm in 25 mlof YPD medium supplemented with 10 mM uridine for 17 hours. Mycelia werecollected by filtration using Millipore's Vacuum Driven DisposableFiltration System (Millipore, Bedford, Mass.) and washed twice withdeionized water and twice with 1.2 M sorbitol. Protoplasts weregenerated by suspending the washed mycelia in 20 ml of 1.2 M sorbitolcontaining 15 mg of Glucanex per ml and 0.36 units of chitinase per mland incubating for 15-25 minutes at 34° C. with gentle shaking at 90rpm. Protoplasts were collected by centrifuging for 7 minutes at 400×gand washed twice with ice cold 1.2 M sorbitol. The protoplasts werecounted using a hemacytometer and re-suspended in STC to a finalconcentration of 1×10⁸ protoplasts per ml. Excess protoplasts werestored in a Cryo 1° C. Freezing Container (Nalgene, Rochester, N.Y.) at−80° C.

Approximately 1 μg of Pme I digested pCW045 was added to 100 μl ofprotoplast solution and mixed gently, followed by 260 μl of PEG buffer,mixed, and incubated at room temperature for 30 minutes. STC (3 ml) wasthen added, mixed and the transformation solution was plated onto COVEplates using Aspergillus nidulans amdS selection. The plates wereincubated at 28° C. for 5-7 days. Transformants were sub-cultured ontoCOVE2 plus uridine plates and grown at 28° C. for 7 days.

The Trichoderma reesei transformants were cultivated in 125 ml baffledshake flasks containing 25 ml of cellulase-inducing media at pH 6.0inoculated with spores of the transformants and incubated at 28° C. and200 rpm for 7 days. Trichoderma reesei RutC30 was run as a control.Culture broth samples were removed at day 7. SDS-PAGE was carried outusing Criterion Tris-HCl (8-16% polyacrylamide) gels (BioRad, Hercules,Calif.) with the Criterion SDS-PAGE System (BioRad, Hercules, Calif.).Five μl of day 7 supernatants were suspended in 5 μl of 2× Sample Buffer(BioRad, Hercules, Calif.) and heated in the presence of 1%beta-mercaptoethanol for 5 minutes. The supernatant samples were loadedonto a polyacrylamide gel and subjected to electrophoresis with 1×Tris/Glycine/SDS as running buffer (BioRad, Hercules, Calif.). Theresulting gel was stained with Bio-Safe Coomassie Stain (BioRad,Hercules, Calif.). Candidate transformants were evaluated for theirability to express the variant CeI7A cellobiohydrolase I 776-M57 atlevels that were apparently equivalent to levels observed in theTrichoderma reesei parent strain. A single transformant was selected onthis basis for larger scale production in fermentation.

Fermentation was performed using the strain expressing cellobiohydrolaseI variant 776-M57, and the Trichoderma reesei RutC30 (host strain) wasrun as a control. Spores of Trichoderma reesei RutC30 were inoculatedinto 500 ml shake flasks, containing 100 ml of Trichoderma InoculumMedium. The flasks were placed into an orbital shaker at 28° C. forapproximately 48 hours at which time 50 ml of the culture was inoculatedinto 1.8 liters of Trichoderma Fermentation Medium in a 2 literfermentation vessel. The fermentations were run at pH 5.0, 28° C., withminimum dissolved oxygen at 25% at a 1.0 VVM air flow and an agitationof 1100. Trichoderma Feed Medium was administrated into the fermentationvessel at 18 hours with a feed rate of 3.6 g/hour for 33 hours and then7.2 g/hour. The fermentations ran for 165 hours at which time the finalfermentation broths were centrifuged and the supernatants stored at −20°C.

Example 18 Identification of a Glycosyl Hydrolase Family GH3A Gene inthe Genomic Sequence of Aspergillus fumigatus

A tblastn search (Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402) of the Aspergillus fumigatus partial genome sequence (TheInstitute for Genomic Research, Rockville, Md.) was carried out using asquery a beta-glucosidase protein sequence from Aspergillus aculeatus(Accession No. P48825). Several genes were identified as putative FamilyGH3A homologs based upon a high degree of similarity to the querysequence at the amino acid level. One genomic region of approximately3000 by with greater than 70% identity to the query sequence at theamino acid level was chosen for further study.

Example 19 Aspergillus fumigatus Genomic DNA Extraction

Aspergillus fumigatus was grown in 250 ml of potato dextrose medium in abaffled shake flask at 37° C. and 240 rpm. Mycelia were harvested byfiltration, washed twice in TE (10 mM Tris-1 mM EDTA) and frozen underliquid nitrogen. Frozen mycelia were ground, by mortar and pestle, to afine powder, which was resuspended in pH 8.0 buffer containing 10 mMTris, 100 mM EDTA, 1% Triton X-100, 0.5 M guanidine-HCl, and 200 mMNaCl. DNase free RNase A was added at a concentration of 20 mg/liter andthe lysate was incubated at 37° C. for 30 minutes. Cellular debris wasremoved by centrifugation, and DNA was isolated by using a Qiagen Maxi500 column (QIAGEN Inc., Valencia, Calif.). The columns wereequilibrated in 10 ml of QBT washed with 30 ml of QC, and eluted with 15ml of QF (all buffers from QIAGEN Inc., Valencia, Calif.). DNA wasprecipitated in isopropanol, washed in 70% ethanol, and recovered bycentrifugation. The DNA was resuspended in TE buffer.

Example 20 Cloning of the Family GH3A Beta-Glucosidase Gene andConstruction of an Aspergillus oryzae Expression Vector

Two synthetic oligonucleotide primers shown below were designed to PCRamplify a Aspergillus fumigatus gene encoding a putative Family GH3Abeta-glucosidase from the genomic DNA prepared in Example 19. AnInFusion Cloning Kit (BD Biosciences, Palo Alto, Calif.) was used toclone the fragment directly into the expression vector, pAlLo2 (Example8; FIG. 5).

Forward primer: (SEQ ID NO: 54) 5′-ACTGGATTTACCATGAGATTCGGTTGGCTCG-3′Reverse primer: (SEQ ID NO: 55) 5′-AGTCACCTCTAGTTACTAGTAGACACGGGGC-3′Bold letters represent coding sequence. The remaining sequence containssequence identity compared with the insertion sites of pAlLo2 (FIG. 5).

Fifty picomoles of each of the primers above were used in a PCR reactioncontaining 100 ng of Aspergillus fumigatus genomic DNA, 1×PfxAmplification Buffer (Invitrogen, Carlsbad, Calif.), 1.5 μl of 10 mMblend of dATP, dTTP, dGTP, and dCTP, 2.5 units of Platinum Pfx DNAPolymerase (Invitrogen, Carlsbad, Calif.), 1 μl of 50 mM MgSO₄ and 2.5μl of 10×pCRx Enhancer solution (Invitrogen, Carlsbad, Calif.) in afinal volume of 50 μl. The amplification conditions were one cycle at94° C. for 2 minutes; 30 cycles each at 94° C. for 15 seconds, 55° C.for 30 seconds, and 68° C. for 3 minutes. The heat block then went to a4° C. soak cycle.

The reaction products were isolated on a 1.0% agarose gel using 40 mMTris base-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer where a 3kb product band was excised from the gel and purified using a QIAquickGel Extraction Kit (QIAGEN, Valencia, Calif.) according to themanufacturer's instructions.

The fragment was then cloned into the pAlLo2 expression vector using anInfusion Cloning Kit. The vector was digested with restrictionendonucleases Nco I and Pac I (using conditions specified by themanufacturer). The fragment was purified by gel electrophoresis andQiaquick gel purification. The gene fragment and the cut vector wereligated together in a reaction resulting in the expression plasmidpEJG97 (FIG. 14) in which transcription of the Family GH3Abeta-glucosidase gene was under the control of the NA2-tpi promoter. Thereaction (50 μl) was composed of 1×InFusion Buffer (BD Biosciences, PaloAlto, Calif.), 1×BSA (BD Biosciences, Palo Alto, Calif.), 1 μl ofInfusion enzyme (diluted 1:10) (BD Biosciences, Palo Alto, Calif.), 150ng of pAlLo2 digested with Nco I and Pac I, and 50 ng of the Aspergillusfumigatus beta-glucosidase purified PCR product. The reaction wasincubated at room temperature for 30 minutes. One μl of the reaction wasused to transform E. coli XL10 Solopac Gold cells (Stratagene, La Jolla,Calif.). An E. coli transformant containing the pEJG97 plasmid wasdetected by restriction digestion and plasmid DNA was prepared using aBioRobot 9600 (QIAGEN, Inc., Valencia, Calif.).

Example 21 Characterization of the Aspergillus fumigatus GenomicSequence Encoding a Family GH3A Beta-Glucosidase

DNA sequencing of the Aspergillus fumigatus beta-glucosidase gene frompEJG97 was performed with a Perkin-Elmer Applied Biosystems Model 377 XLAutomated DNA Sequencer (Perkin-Elmer/Applied Biosystems, Inc., FosterCity, Calif.) using dye-terminator chemistry (Giesecke et al., 1992,Journal of Virology Methods 38: 47-60) and primer walking strategy.Nucleotide sequence data were scrutinized for quality and all sequenceswere compared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash.).

A gene model for the Aspergillus fumigatus sequence was constructedbased on similarity to homologous genes from Aspergillus aculeatus,Aspergillus niger, and Aspergillus kawachii. The nucleotide sequence(SEQ ID NO: 56) and deduced amino acid sequence (SEQ ID NO: 57) areshown in FIG. 15. The genomic fragment encodes a polypeptide of 863amino acids, interrupted by 8 introns of 62, 55, 58, 63, 58, 58, 63 and51 bp. The % G+C content of the gene is 54.3%. Using the SignalPsoftware program (Nielsen et al., 1997, Protein Engineering 10: 1-6), asignal peptide of 19 residues was predicted. The predicted matureprotein contains 844 amino acids with a molecular mass of 91.7 kDa.

A comparative alignment of beta-glucosidase sequences was determinedusing the Clustal W method (Higgins, 1989, CABIOS 5: 151-153) using theLASERGENE™ MEGALIGN™ software (DNASTAR, Inc., Madison, Wis.) with anidentity table and the following multiple alignment parameters: Gappenalty of 10 and gap length penalty of 10. Pairwise alignmentparameters were Ktuple=1, gap penalty=3, windows=5, and diagonals=5. Thealignment showed that the deduced amino acid sequence of the Aspergillusfumigatus beta-glucosidase gene shared 78%, 76%, and 76% identity to thededuced amino acid sequences of the Aspergillus aculeatus (accessionnumber P48825), Aspergillus niger (000089), and Aspergillus kawachii(P87076) beta-glucosidases.

Example 22 Expression of the Aspergillus fumigatus Family GH3ABeta-Glucosidase Gene in Aspergillus oryzae JAL250

Aspergillus oryzae Ja1250 protoplasts were prepared according to themethod of Christensen et al., 1988, Bio/Technology 6: 1419-1422. Five μgof pEJG97 (as well as pAlLo2 as a vector control) was used to transformAspergillus oryzae JAL250.

The transformation of Aspergillus oryzae Ja1250 with pEJG97 yieldedabout 100 transformants. Ten transformants were isolated to individualPDA plates.

Confluent PDA plates of five of the ten transformants were washed with 5ml of 0.01% Tween 20 and inoculated separately into 25 ml of MDU2BPmedium in 125 ml glass shake flasks and incubated at 34° C., 250 rpm.Five days after incubation, 0.5 μl of supernatant from each culture wasanalyzed using 8-16% Tris-Glycine SDS-PAGE gels (Invitrogen, Carlsbad,Calif.) according to the manufacturer's instructions. SDS-PAGE profilesof the cultures showed that one of the transformants (designatedtransformant 1) had a major band of approximately 130 kDa.

A confluent plate of transformant 1 (grown on PDA) was washed with 10 mlof 0.01% Tween 20 and inoculated into a 2 liter Fernbach containing 400ml of MDU2BP medium to generate broth for characterization of theenzyme. The flask was harvested on day 5 and filtered using a 0.22 μm GPExpress plus Membrane (Millipore, Bedford, Mass.).

A single transformant of pEJG97 in Aspergillus oryzae was grown in afermentation tank. Spores of Aspergillus oryzae were inoculated into 500ml shake flasks, containing 100 ml of Inoculum Medium. The flasks wereplaced into an orbital shaker at 34° C., at 200 rpm for approximately 24hours at which time 50 ml of the culture was inoculated into 1.8 litersof Fermentation Medium in a 2 liter fermentation vessel. Thefermentations were run at pH 7.0, 34° C., with minimum dissolved oxygenat 25% at a 1.0 VVM air flow and an agitation of 1100. Feed Medium wasadministrated into the fermentation vessel at the dissolved oxygen spikewith a feed rate of 4 g/hour. The fermentations ran for 164 hours atwhich time the final fermentation broths were centrifuged and thesupernatants stored at −20° C.

Example 23 Characterization of Aspergillus fumigatus Beta-Glucosidase

A 3 ml aliquot of the Aspergillus fumigatus beta-glucosidase, obtainedas described in Example 22, was desalted using a BioRad Econo-Pac 10DGdesalting column (BioRad, Hercules, Calif.), resulting in approx. 4 mlof desalted broth in 100 mM sodium citrate pH 5.0. The desalted brothwas then concentrated to approximately 180 μl using an Amicon CentriconPlus-20 (Biomax-5, 5 kDa cutoff, PES membrane) and diluted with 100 mMsodium citrate pH 5.0 to a final volume of approximately 500 μl. BCAassay (Smith et al., 1985, Anal. Biochem. 150: 76-85) of the desalted,concentrated broth showed a concentration of 1.00 mg of protein per ml.A second aliquot was also desalted to obtain more material, whichyielded similar results.

SDS-PAGE (BioRad Criterion 7.5% Tris-HCl) of the concentrated desaltedsamples showed a major band at approx. 130 kD. The major band at 130 kDwas cut out of the gel and submitted to N-terminal amino acidsequencing.

The Aspergillus fumigatus beta-glucosidase from thedesalted/concentrated broth was evaluated for thermal stability at 50°,65°, and 70° C. Assay conditions at 50° and 65° C. were: 100 mM sodiumcitrate pH 5.0, 0.01% Tween-20, 4 mMp-nitrophenyl-beta-D-glucopyranoside, [protein]_(AfumGH3A)=6.9×10⁻⁶mg/ml, incubated at 50° and 65° C. Aliquots were taken at 0.5, 1, 2, 3,3.75, and 24 hours. To each aliquot was added 1 M sodium carbonate pH10.0, and the p-nitrophenyl anion concentration was determined from theabsorbance at 405 nm. At 70° C., the assay conditions were: 100 mMsodium citrate (pH 5.0), 0.01% Tween-20, 4 mMp-nitrophenyl-beta-D-glucopyranoside, [protein]=5.6×10⁻⁶ mg/ml. Aliquotswere taken at 0.25, 0.5, 1, 2, 4, and 5 hours. To each aliquot was added1 M sodium carbonate pH 10.0, and the p-nitrophenyl anion concentrationwas determined from the absorbance at 405 nm. Note that each of theabove protein concentrations refers to total protein concentration inthe assay, as they were all assayed as broths rather than purifiedenzymes.

The results are shown in FIGS. 16 and 17. Thermal stability was definedas linear reaction kinetics for a given time interval, within areasonable percent conversion (<15%) to p-nitrophenyl anion. TheAspergillus fumigatus beta-glucosidase appeared to be stable at least 24hours at 50° C., although there were no time points between 4 hours and24 hours. At 65° C. it appeared to be stable at least 3.75 hours, butafterward the stability gradually decreased with a 65% conversion top-nitrophenyl anion at 24 hours. This conversion was quite high, so someof the observed decrease in rate may have been due to depletion ofsubstrate and/or product inhibition in addition to possible thermalinactivation. At 70° C. it appeared to be stable for 1 hour, andreasonably stable to 2 hours.

Broth containing Family GH3A beta-glucosidase from Aspergillusfumigatus, obtained as described in Example 22, was first desalted(BioRad Econo-Pac 10DG column) and concentrated (Centricon Plus-20,Biomax-5, 5 kD cut-off), to a concentration of 0.92 mg/ml (BCA assay).Then, it was incubated at 0.037 and 0.0092 μg/ml total protein with 10mM cellobiose in 100 mM sodium citrate pH 5.0 plus 0.01% Tween-20 at 65°C. Aliquots were taken at 0.5, 1, 2, 3, 4, 6, and 19 hours. Aliquotswere boiled 6 minutes to terminate the reaction, and the glucoseconcentration determined using the Trinder assay (Sigma Chemical Co.,St. Louis, Mo.) and external glucose standards. Results are shown inFIG. 18. The beta-glucosidase appeared to maintain 90% of its activityto 6 hours, and 65% to 19 hours at 65° C. at the lower protein loading(0.0092 μg/ml). The beta-glucosidase appeared to be reasonably stable upto 6 hours at 65° C.

Example 24 Assay of CeI7A Variant 776-M57 in Combination withTrichoderma Cellulases

Pretreated corn stover (PCS) was obtained from U.S. Department of EnergyNational Renewable Energy Laboratory (NREL). The water-insoluble solidsin PCS include: 56.5% cellulose, 4.6% hemicellulose, and 28.4% lignin.Pretreatment conditions were: corn stover, 1.4% (wt/vol) sulfuric acid,165° C., 107 p.s.i., for 8 minutes. Prior to assay, PCS was washed witha large volume of distilled deionized water on a glass filter. PCS wasthen milled using a coffee grinder to reduce particle size, then washedfurther with water on a 22 μm Millipore filter (6P Express Membrane,Stericup, Millipore, Billerica, Mass.). The washed PCS was resuspendedin deionized water to make a 20 mg/ml suspension, and stored at 4° C.

Trichoderma reesei fermentation broths were filtered (0.4 μm Membrane,Stericup, Millipore, Billerica, Mass.), and protein content was assayedby BCA assay (Pierce Biotech., Inc., Rockford, Ill.). The thermostablebeta-glucosidase from Aspergillus fumigatus was used in the PCS assays.The Aspergillus fumigatus beta-glucosidase was prepared as described inExample 22. Fermentation broths of the heterologously expressedAspergillus fumigatus beta-glucosidase were filtered (0.4 μm Membrane,Stericup, Millipore, Billerica, Mass.), and protein content was assayedby BCA assay (Pierce, Biotech., Inc., Rockford, Ill.). PCS assays werecarried out in a volume of 1.0 ml. Assays included 10 mg of PCS per ml,50 mM sodium acetate buffer (pH 5.0), 8.5 mg of total fermentation brothprotein per gram of cellulose, and 0.27 mg of Aspergillus fumigatusbeta-glucosidase fermentation broth. Assays were maintained at 55° C.and at 60° C. in sealed assay tubes (ImmunoWare Microtubes, PiereceBiotech., Inc., Rockford, Ill.) for 5 days with intermittent inversionof assay tubes.

Approximately every 24 hours, time points were taken from the PCSreactions. Ten microliter aliquots were removed from the assay tubesinto 90 μl of alkaline quench mixture (0.102 M Na₂CO₃ plus 0.058 MNaHCO₃). Dilution of the quenched samples to one-eighth or one-sixteenthof the starting concentration was performed. The concentration ofreducing sugars in samples produced by degradation of PCS was determinedby p-hydroxybenzoic acid hydrazide (PHBAH) assay (Lever, 1972, AnalBiochem 47: 273-279) using 2 parts of 1.25% PHBAH in alkaline quenchmixture added to 3 parts of the quenched assay solution. This solutionwas then heated for 10 minutes at 95° C., samples were then diluted intowater, and absorbance was measured at 405 nm using a UltraMark PlateReader (Bio-Rad, Hercules, Calif.). The absorbance at 405 nm wasconverted into glucose equivalents using a glucose standard curve.Finally, the degree of cellulose conversion was calculated, using theinitial concentration of cellulose, and a weight gain factor inconverting cellulose to glucose. The degree of cellulose conversion toreducing sugar (RS yield, %) was calculated using the followingequation:

$\begin{matrix}{{{RS}\mspace{14mu} {Yield}_{(\%)}} = {{RS}_{({{mg}\text{/}{ml}})}*100*162\text{/}\left( {5.65_{({{mg}\text{/}{ml}})}*180} \right)}} \\{= {{RS}_{({{mg}\text{/}{ml}})}*100\text{/}\left( {5.65_{({{mg}\text{/}{ml}})}*1.111} \right)}}\end{matrix}$

FIG. 19 shows the time course profiles of PCS hydrolysis by the parentTrichoderma reesei strain, and the strain which expresses variant776-M57 in place of the wild type CeI7A cellobiohydrolase I. It isapparent that the strain containing the variant outperforms the parentunder these conditions.

Deposit of Biological Materials

The following biological materials have been deposited under the termsof the Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession numbers:

Deposit Accession Number Date of Deposit pAJO52 NRRL B-30683 Jul. 29,2003 776-M1 NRRL B-30657 May 22, 2003 776-M4 NRRL B-30658 May 22, 2003776-M23 NRRL B-30659 May 22, 2003 776-M26 NRRL B-30661 May 22, 2003776-M32 NRRL B-30662 May 22, 2003 776-M53 NRRL B-30663 May 22, 2003776-M57 NRRL B-30664 May 22, 2003 776-M108 NRRL B-30665 May 22, 2003776-M109 NRRL B-30666 May 22, 2003 776-M21 NRRL B-30674 Jul. 28, 2003776-M22 NRRL B-30675 Jul. 28, 2003 776-M41 NRRL B-30676 Jul. 28, 2003776-M42 NRRL B-30677 Jul. 28, 2003 776-M52 NRRL B-30678 Jul. 28, 2003776-M71 NRRL B-30679 Jul. 28, 2003 776-M73 NRRL B-30680 Jul. 28, 2003776-M124 NRRL B-30681 Jul. 28, 2003 776-M125 NRRL B-30682 Jul. 28, 2003776-M273 NRRL B-30762 Aug. 25, 2004

The strains have been deposited under conditions that assure that accessto the cultures will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposits represent substantially pure cultures of thedeposited strains. The deposits are available as required by foreignpatent laws in countries wherein counterparts of the subjectapplication, or its progeny are filed. However, it should be understoodthat the availability of a deposit does not constitute a license topractice the subject invention in derogation of patent rights granted bygovernmental action.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1-441. (canceled)
 442. A variant of a parent glycoside hydrolase,comprising a substitution at one or more positions corresponding topositions 21, 94, 157, 205, 206, 247, 337, 350, 373, 383, 438, 455, 467,and 486 of amino acids 1 to 513 of SEQ ID NO: 2, and optionally furthercomprising a substitution at one or more positions corresponding topositions 8, 22, 41, 49, 57, 113, 193, 196, 226, 227, 246, 251, 255,259, 301, 356, 371, 411, and 462 of amino acids 1 to 513 of SEQ ID NO:2, wherein the variant has glycoside hydrolase activity.
 443. Adetergent composition comprising a variant of claim 442 and asurfactant.
 444. A method for degrading cellulose- andhemicellulose-containing biomass, comprising treating the biomass withan effective amount of a variant of claim 442 and recovering thedegraded biomass.
 445. The method of claim 444, further comprisingtreating the biomass with an effective amount of endo-1,4-beta-glucanaseand exo-1,4-beta-D-glucanase.